Peptidomimetic macrocycles and formulations thereof

ABSTRACT

Aqueous pharmaceutical formulations, for parenteral administration, comprising peptidomimetic macrocycle or a pharmaceutically acceptable salt thereof wherein the peptidomimetic macrocycle binds to MDM2 and/or MDMX proteins are disclosed. Also disclosed are methods of treating diseases and disorders using the aqueous pharmaceutical formulations disclosed herein.

CROSS REFERENCE

This application is a Continuation application of U.S. application Ser.No. 17/091,267 filed Nov. 6, 2020, which application is a Continuationapplication of U.S. application Ser. No. 14/864,687 filed Sep. 24, 2015,now U.S. Pat. No. 10,905,739, which application claims priority to U.S.Provisional Application No. 62/054,842, filed Sep. 24, 2014, each ofwhich is incorporated herein by reference in its entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jun. 4, 2021, isnamed 35224-803.301_SL.txt and is 1,382,300 bytes in size.

BACKGROUND OF THE DISCLOSURE

The human transcription factor protein p53 induces cell cycle arrest andapoptosis in response to DNA damage and cellular stress, and therebyplays a critical role in protecting cells from malignant transformation.The E3 ubiquitin ligase MDM2 (also known as HDM2 or human double minute2) negatively regulates p53 function through a direct bindinginteraction that neutralizes the p53 transactivation activity, leads toexport from the nucleus of p53 protein, and targets p53 for degradationvia the ubiquitylation-proteasomal pathway. Loss of p53 activity, eitherby deletion, mutation, or MDM2 overexpression, is the most common defectin human cancers. Tumors that express wild type p53 are vulnerable topharmacologic agents that stabilize or increase the concentration ofactive p53. In this context, inhibition of the activities of MDM2 hasemerged as a validated approach to restore p53 activity and resensitizecancer cells to apoptosis in vitro and in vivo. MDMX (also known asMDM4, HDM4 or human double minute 4) has more recently been identifiedas a similar negative regulator of p53, and studies have revealedsignificant structural homology between the p53 binding interfaces ofMDM2 and MDMX. MDMX has also been observed to be overexpressed in humantumors. The p53-MDM2 and p53-MDMX protein-protein interactions aremediated by the same 15-residue alpha-helical transactivation domain ofp53, which inserts into hydrophobic clefts on the surface of MDM2 andMDMX. Three residues within this domain of wild type p53 (F19, W23, andL26) are essential for binding to MDM2 and MDMX.

There remains a considerable need for compounds capable of binding toand modulating the activity of p53, MDM2 and/or MDMX. Provided hereinare aqueous pharmaceutical formulations comprising p53-basedpeptidomimetic macrocycles that modulate an activity of p53. Alsoprovided herein are aqueous pharmaceutical formulations comprisingp53-based peptidomimetic macrocycles that inhibit the interactionsbetween p53, MDM2 and/or MDMX proteins. Further, provided herein areaqueous pharmaceutical formulations comprising p53-based peptidomimeticmacrocycles that can be used for treating diseases including but notlimited to cancer and other hyperproliferative diseases.

SUMMARY OF THE DISCLOSURE

In one aspect, the disclosure provides an aqueous pharmaceuticalformulation comprising a peptidomimetic macrocycle that binds to MDM2and/or MDMX proteins or a pharmaceutically acceptable salt thereof, abuffering agent, a tonicity agent, and a stabilizing agent wherein theamount of the peptidomimetic macrocycle in the aqueous pharmaceuticalformulation is equal to or greater than 15 mg/mL and wherein the aqueouspharmaceutical formulation comprises less than 2% w/v of any micelleforming agent. The micelle forming agent can be solutol-HS-15. In someexamples, the peptidomimetic macrocycle forms a micelle in absence of asurfactant.

In another aspect, the disclosure provides an aqueous pharmaceuticalformulation comprising (i) a peptidomimetic macrocycle or apharmaceutically acceptable salt thereof, wherein the amount of thepeptidomimetic macrocycle in the aqueous pharmaceutical formulation isequal to or greater than 15 mg/mL; (ii) a buffering agent; (iii) astabilizing agent; and (iv) a tonicity agent, wherein the molar ratio ofthe peptidomimetic macrocycle to the buffering agent is in the range of0.01-2.5.

In another aspect, the disclosure provides an aqueous pharmaceuticalformulation comprising a peptidomimetic macrocycle that binds to atarget protein with a K_(D) value of 1×10⁻⁷ M or less, or apharmaceutically acceptable salt thereof, a buffering agent, a tonicityagent, and a stabilizing agent wherein the amount of the peptidomimeticmacrocycle in the aqueous pharmaceutical formulation is equal to orgreater than 15 mg/mL and wherein the aqueous pharmaceutical formulationcomprises less than 2% w/v of any micelle forming agent, wherein thepeptidomimetic macrocycle or pharmaceutically acceptable salt thereofhas: (a) a length value of from 10 to 24 amino acids, (b) a von Heijnevalue of from 2 to 10, (c) a net charge of from −4 to +2, (d) a percentalanine content of from 15% to 50%, (e) or any combination of (a)-(d).

In some embodiments, the peptidomimetic macrocycle or pharmaceuticallyacceptable salt thereof is not precipitated in the formulation. In someembodiments, an aqueous solubility of the peptidomimetic macrocycle isdetermined by evaluating the turbidity of a solution comprising thepeptidomimetic macrocycle. In some embodiments, the peptidomimeticmacrocycle or pharmaceutically acceptable salt thereof has anamphipathicity that falls in a range that is optimal for cellpermeability.

In some embodiments, the peptidomimetic macrocycle or pharmaceuticallyacceptable salt thereof has a length value of from 14 to 20 amino acids.In some embodiments, the peptidomimetic macrocycle or pharmaceuticallyacceptable salt thereof has a von Heijne value of from 2 to 9. In someembodiments, the peptidomimetic macrocycle or pharmaceuticallyacceptable salt thereof has a von Heijne value of from 3 to 8. In someembodiments, the peptidomimetic macrocycle or pharmaceuticallyacceptable salt thereof has a von Heijne value of from 4 to 7. In someembodiments, the peptidomimetic macrocycle or pharmaceuticallyacceptable salt thereof has a net charge of from −2 to 0. In someembodiments, the peptidomimetic macrocycle or pharmaceuticallyacceptable salt thereof has a percent alanine content of from 15% to40%. In some embodiments, the peptidomimetic macrocycle orpharmaceutically acceptable salt thereof has a percent alanine contentof from 20% to 40%. In some embodiments, the peptidomimetic macrocycleor pharmaceutically acceptable salt thereof has a percent alaninecontent of from 25% to 40%. In some embodiments, the peptidomimeticmacrocycle or pharmaceutically acceptable salt thereof has a lengthvalue of from 14 to 20 amino acids, a von Heijne value of from 4 to 7, anet charge of from −2 to 0, and a percent alanine content of from 25% to40%. In some embodiments, the peptidomimetic macrocycle orpharmaceutically acceptable salt thereof is soluble, does not haveoff-target effects, or a combination thereof.

In some embodiments, the peptidomimetic macrocycle or pharmaceuticallyacceptable salt thereof comprises a first C-terminal amino acid that ishydrophobic. In some embodiments, the peptidomimetic macrocycle orpharmaceutically acceptable salt thereof comprises a second C-terminalamino acid that is hydrophobic. In some embodiments, the peptidomimeticmacrocycle or pharmaceutically acceptable salt thereof comprises a thirdC-terminal amino acid that is hydrophobic. In some embodiments, thepeptidomimetic macrocycle or pharmaceutically acceptable salt thereofcomprises a fourth C-terminal amino acid that is hydrophobic. In someembodiments, the peptidomimetic macrocycle or pharmaceuticallyacceptable salt thereof comprises a fifth C-terminal amino acid that ishydrophobic. In some embodiments, the peptidomimetic macrocycle orpharmaceutically acceptable salt thereof comprises a sixth C-terminalamino acid that is hydrophobic.

In some embodiments, the first amino acid connected to the crosslinkeris N-terminal to the second amino acid connected to the crosslinker, andwherein the peptidomimetic macrocycle or pharmaceutically acceptablesalt thereof comprises 1, 2, 3, 4, 5, 6, 7, or 8 amino acids that areC-terminal to the second amino acid connected to the crosslinker.

In some embodiments, the first amino acid connected to the crosslinkeris N-terminal to the second amino acid connected to the crosslinker, andwherein the peptidomimetic macrocycle or pharmaceutically acceptablesalt thereof comprises 1, 2, 3, 4, 5, or 6 hydrophobic amino acids thatare C-terminal to the second amino acid connected to the crosslinker.

In some embodiments, the first amino acid connected to the crosslinkeris N-terminal to the second amino acid connected to the crosslinker, andwherein the peptidomimetic macrocycle or pharmaceutically acceptablesalt thereof comprises 1, 2, or 3 glutamines that are C-terminal to thesecond amino acid connected to the crosslinker.

In some embodiments, the amino acid that is hydrophobic is a smallhydrophobic amino acid. In some embodiments, the amino acid that ishydrophobic is an alanine, a D-alanine, or an Aib.

In some embodiments, the peptidomimetic macrocycle or pharmaceuticallyacceptable salt thereof is a helical polypeptide. In some embodiments,the peptidomimetic macrocycle or pharmaceutically acceptable saltthereof comprises an α-helix. In some embodiments, the peptidomimeticmacrocycle or pharmaceutically acceptable salt thereof comprises anamphipathic α-helix.

In some embodiments, the first amino acid connected to the crosslinkeror the second amino acid connected to the crosslinker is anα,α-disubstituted amino acid. In some embodiments, the first amino acidconnected to the crosslinker and the second amino acid connected to thecrosslinker are α,α-disubstituted amino acids. In some embodiments, thefirst amino acid connected to the crosslinker and the second amino acidconnected to the crosslinker are separated by two amino acids. In someembodiments, the first amino acid connected to the crosslinker and thesecond amino acid connected to the crosslinker are separated by threeamino acids. In some embodiments, the first amino acid connected to thecrosslinker and the second amino acid connected to the crosslinker areseparated by six amino acids. In some embodiments, the crosslinker spans1 turn of an α-helix of the peptidomimetic macrocycle. In someembodiments, the crosslinker spans 2 turns of an α-helix of thepeptidomimetic macrocycle. In some embodiments, the length of thecrosslinker is from about 5 Å to about 9 Å per turn of an α-helix of thepeptidomimetic macrocycle. In some embodiments, the peptidomimeticmacrocycle or pharmaceutically acceptable salt thereof provides atherapeutic effect. In some embodiments, an ability of thepeptidomimetic macrocycle or pharmaceutically acceptable salt thereof topenetrate cell membranes by an energy-dependent process is improvedrelative to a corresponding uncrosslinked peptidomimetic macrocycle. Insome embodiments, the ability of the peptidomimetic macrocycle orpharmaceutically acceptable salt thereof to penetrate cell membranes byan energy-independent process is improved relative to a correspondinguncrosslinked peptidomimetic macrocycle. In some embodiments, theenergy-dependent process is primary active transport, secondarytransport, endocytosis, or a combination thereof. In some embodiments,the energy-dependent process is active transport. In some embodiments,the energy-independent process is passive diffusion, facilitateddiffusion, filtration, or a combination thereof. In some embodiments,the energy-independent process is passive transport.

In some embodiments, the peptidomimetic macrocycle or pharmaceuticallyacceptable salt thereof binds to HDM2 with a K_(D) value of 1×10⁻⁷ M orless. In some embodiments, the peptidomimetic macrocycle orpharmaceutically acceptable salt thereof binds to HDM2 or HDM4 with aK_(D) value of 1×10⁻⁷ M or less. In some embodiments, the peptidomimeticmacrocycle or pharmaceutically acceptable salt thereof binds to HDM4with a K_(D) value of 1×10⁻⁷ M or less. In some embodiments, thepeptidomimetic macrocycle or pharmaceutically acceptable salt thereofbinds to a PB1 peptide binding site of a PA protein with a K_(D) valueof 1×10⁻⁷ M or less. In some embodiments, the peptidomimetic macrocycleor pharmaceutically acceptable salt thereof binds to a PB2 peptidebinding site of a PB1 protein with a K_(D) value of 1×10⁻⁷ M or less. Insome embodiments, the peptidomimetic macrocycle or pharmaceuticallyacceptable salt thereof binds to viral polymerase, for example, aRNA-dependent RNA polymerase with a K_(D) value of 1×10⁻⁷ M or less. Insome embodiments, the peptidomimetic macrocycle or pharmaceuticallyacceptable salt thereof inhibits an influenza RNA-dependent RNApolymerase. In some embodiments, the virus is influenza virus. In someembodiments, the peptidomimetic macrocycle is capable of competing withthe binding of a peptide of the sequence MDVNPTLLFLKVPAQ (SEQ ID NO: 1)or MERIKELRNLM (SEQ ID NO: 2) to the viral RNA-dependent RNA polymerase.In some embodiments, the peptidomimetic macrocycle or pharmaceuticallyacceptable salt thereof binds to MCL-1, BCL-X_(L), BCL-2, or acombination thereof with a K_(D) value of 1×10⁻⁷ M or less. In someembodiments, the peptidomimetic macrocycle or pharmaceuticallyacceptable salt thereof binds to MCL-1 with a K_(D) value of 1×10⁷ M orless. In some embodiments, the peptidomimetic macrocycle orpharmaceutically acceptable salt thereof binds to BCL-X_(L) with a K_(D)value of 1×10⁻⁷ M or less. In some embodiments, the peptidomimeticmacrocycle or pharmaceutically acceptable salt thereof binds to BCL-2with a K_(D) value of 1×10⁻⁷ M or less.

In some embodiments, the peptidomimetic macrocycle or pharmaceuticallyacceptable salt thereof has an IC₅₀ value of 100 nM or less to a targetprotein. In some embodiments, the peptidomimetic macrocycle orpharmaceutically acceptable salt thereof has an EC₅₀ value of 100 μM orless.

In some embodiments, the peptidomimetic macrocycle or pharmaceuticallyacceptable salt thereof has an IC₅₀ value of 10 nM or less to a targetprotein. In some embodiments, the peptidomimetic macrocycle orpharmaceutically acceptable salt thereof has an EC₅₀ value of 10 μM orless. In some embodiments, the peptidomimetic macrocycle orpharmaceutically acceptable salt thereof has an IC₅₀ value of 1 nM orless to a target protein. In some embodiments, the peptidomimeticmacrocycle or pharmaceutically acceptable salt thereof has an EC₅₀ valueof 1 μM or less.

In some embodiments, the peptidomimetic macrocycle or pharmaceuticallyacceptable salt thereof has a penetration efficiency value of 100 orless. In some embodiments, the peptidomimetic macrocycle orpharmaceutically acceptable salt thereof has a penetration efficiencyvalue of 10 or less. In some embodiments, the peptidomimetic macrocycleor pharmaceutically acceptable salt thereof has a penetration efficiencyvalue of 1 or less.

In some embodiments, the peptidomimetic macrocycle penetrates cellmembranes by an energy-dependent process and binds to an intracellulartarget with a K_(D) value of 1×10⁻⁷ M or less. In some embodiments, theenergy-dependent process comprises primary active transport, secondarytransport, or endocytosis. In some embodiments, the energy-dependentprocess comprises active transport. In some embodiments, thepeptidomimetic macrocycle penetrates cell membranes by anenergy-independent process and binds to an intracellular target with aK_(D) value of 1×10⁻⁷ M or less. In some embodiments, theenergy-independent process comprises passive diffusion, facilitateddiffusion, or filtration. In some embodiments, the energy-independentprocess comprises passive transport.

In some embodiments, the amount of the buffering agent in the aqueouspharmaceutical formulations of the disclosure is 0.001-10% w/v, thestabilizing agent in the aqueous pharmaceutical formulations of thedisclosure is 0.001-10% w/v and, the amount of the tonicity agent in theaqueous pharmaceutical formulations of the disclosure 1.0-10% w/v.

The pharmaceutically acceptable salt of the peptidomimetic macrocyclecan be a sodium salt. In some examples, the pharmaceutically acceptablesalt of the peptidomimetic macrocycle can be a potassium, lithium,calcium, zinc or magnesium salt.

Any suitable amount of the peptidomimetic macrocycle can be used in theaqueous pharmaceutical formulations of the disclosure. In some examples,the amount of the peptidomimetic macrocycle present in the aqueouspharmaceutical formulation can be from about 0.1-10% w/v. For example,the amount of the peptidomimetic macrocycle present in the aqueouspharmaceutical formulation can be about 1% w/v, 1.5% w/v, or 2% w/v. Insome examples, the concentration of the peptidomimetic macrocyclepresent in the aqueous pharmaceutical formulation is about 15-100 mg/mL.In some examples, the concentration of the peptidomimetic macrocyclepresent in the aqueous pharmaceutical formulation is about 15-50 mg/mL.In some examples, the concentration of the peptidomimetic macrocyclcpresent in the aqueous pharmaceutical formulation is about 15, 20, 25,or 50 mg/mL.

Any suitable buffering agent can be used in the aqueous pharmaceuticalformulations described herein. In some examples, the buffering agent isselected from a group consisting of ammonia solution, calcium carbonate,tribasic calcium phosphate, citric acid dihydrate, citric acidmonohydrate, dibasic sodium phosphate, diethanolamine, malic acid,monobasic sodium phosphate, monoethanolamine, monosodium glutamate,phosphoric acid, phosphate-citrate buffer (dibasic sodium phosphate andcitric acid), potassium citrate, sodium acetate, sodium bicarbonate,sodium borate, sodium citrate dehydrate, sodium hydroxide, sodiumlactate, sodium carbonate, and triethanolamine(tris(hydroxymethyl)aminomethane). In some examples, the buffering agentis a phosphate buffer. In some examples, the buffering agent is selectedfrom a group consisting of phosphoric acid, dibasic sodium phosphate,monobasic sodium phosphate or a mixture thereof. In some examples, thebuffering agent is 20 mM phosphate buffer

The amount of the buffering agent in the aqueous pharmaceuticalformulations of the disclosure can be from about 0.001-10% w/v. In someexamples, the amount of the buffering agent in the aqueouspharmaceutical formulations of the disclosure is from about 0.01-10%%w/v. In some examples, the amount of the buffering agent in the aqueouspharmaceutical formulations of the disclosure is from about 0.01-5% w/v.In some examples, the amount of the buffering agent in the aqueouspharmaceutical formulations of the disclosure is from about 0.01-1% w/v.In some examples, the amount of the buffering agent present in theaqueous pharmaceutical formulations of the disclosure is about 0.2% w/v.

The stabilizing agent in the aqueous pharmaceutical formulations of thedisclosure can be a non-ionic stabilizing agent. In some examples, thestabilizing agent is a fatty acid ester. In some examples, thestabilizing agent can be a surfactant. In some for examples, thestabilizing agent is a non-ionic surfactant. In some for examples, thestabilizing agent is an anti-oxidant. In some examples the stabilizingagent can be selected from a group consisting of polyoxyethylene glycolalkyl ethers, polyoxypropylene glycol alkyl ethers, glucoside alkylethers, polyoxyethylene glycol octylphenol ethers, polyoxyethyleneglycol alkylphenol ethers, glycerol alkyl esters, polyoxyethylene glycolsorbitan alkyl esters, sorbitan alkyl esters, cocamide MEA, cocamideDEA, dodecyldimethylamine oxide, block copolymers of polyethylene glycoland polypropylene glycol, and polyethoxylated tallow amine. In someexamples, the stabilizing agent can be a polyoxyethylene sorbitan fattyacid ester. In some examples, stabilizing agent can be polysorbate 20,polysorbate 21, polysorbate 40, polysorbate 60, polysorbate 61,polysorbate 65, polysorbate 80, polysorbate 81, polysorbate 85 orpolysorbate 120. In some examples, the stabilizing agent can bepolysorbate 20.

The amount of the stabilizing agent present in the aqueouspharmaceutical formulation is from about 0.001-10% w/v, for example fromabout 0.01-0.05% w/v. In some examples, the amount of the stabilizingagent present in the aqueous pharmaceutical formulations is about 0.03%w/v. In some examples, the aqueous pharmaceutical formulations comprise250-350 ppm polysorbate 20. The aqueous pharmaceutical formulation ofthe disclosure can be a solution. In some examples, the aqueouspharmaceutical formulations can be sterile. In some examples, theaqueous pharmaceutical formulations can be colorless. In some examples,the aqueous pharmaceutical formulations can be a frozen solution. Insome examples, the aqueous pharmaceutical formulation can berefrigerated solution.

In some examples, the aqueous pharmaceutical formulations can beparticulate-free. In some examples, the aqueous pharmaceuticalformulations comprise less than about 6,000 particles of size ≥10 μm inabout 5 mL of the aqueous formulation. In some examples, the aqueouspharmaceutical formulations comprise less than about 600 particles ofsize ≥25 μm in about 5 mL of the aqueous formulation.

In some examples, the aqueous pharmaceutical formulations are dissolvedinto a diluent prior to administration into a subject. The diluent canbe water for injection. In some examples, thee diluent can be solutionof dextrose in water. The amount of the diluent can be from about 50-99%w/v. In some examples, the amount of the diluent can be about 90% w/v.

In some examples, the tonicity agent in the aqueous pharmaceuticalformulations of the disclosure can be a non-ionic tonicity agent. Insome examples, the tonicity agent can be a sugar or a sugar alcohol. Insome examples, the tonicity agent can be a mono- or a disaccharide. Insome cases, the tonicity agent can be selected from a groups consistingof glucose, fructose, galactose, sucrose, lactose, maltose, trehalose,and mixtures thereof.

In some examples, the tonicity agent can be mannitol, glycerin, or acombination thereof. In some examples the tonicity agent can beD-trehalose.

The amount of the tonicity agent present in the aqueous pharmaceuticalformulations can be from about 1-15% w/v. In some examples, the amountof the tonicity agent present in the aqueous pharmaceutical formulationscan be about 8% w/v. The concentration of the tonicity agent can be fromabout 200-300 mM. In some examples, the concentration of the tonicityagent is 240 mM.

The pH of the aqueous pharmaceutical formulations of the disclosure canbe from about 4.0-9.0. In some examples the pH of the aqueouspharmaceutical formulations of the disclosure is from about 4.5-8.5. Insome examples the pH of the aqueous pharmaceutical formulations of thedisclosure is from about 5.0-8.0. In some examples the pH of the aqueouspharmaceutical formulations of the disclosure is from about 5.5-7.5. Insome examples the pH of the aqueous pharmaceutical formulations of thedisclosure is from about 7.0-7.5.

The aqueous pharmaceutical formulations of the disclosure can be stablefor at least two years at a temperature of about −20° C.-25° C. In someexamples, the aqueous pharmaceutical formulations can be stable for atleast one year at a temperature of about −20° C.-25° C. In someexamples, the aqueous pharmaceutical formulations can be stable for atleast 6 months at a temperature of about −20° C.-25° C. In someexamples, the aqueous pharmaceutical formulations can be stable for atleast 3 months at a temperature of about −20° C.-25° C. In someexamples, the aqueous pharmaceutical formulations can be stable for atleast 3 months at a temperature of about 45° C. In some examples, theaqueous pharmaceutical formulations can be stable for at least 6 monthsat a temperature of about 45° C. In some examples, the aqueouspharmaceutical formulations can be stable for at least 3 weeks at atemperature of about 75° C. In some examples, the aqueous pharmaceuticalformulations can be stable for at least 1.5 weeks at a temperature ofabout 75° C.

In some examples, the aqueous pharmaceutical formulations upon storagefor 24 months at from about 2° C.-8° C. can comprise at least 95% of theinitial amount of the peptidomimetic macrocycle. In some examples, theaqueous pharmaceutical formulations upon storage for 12 months at fromabout 2° C.-8° C. can comprise at least 95% of the initial amount of thepeptidomimetic macrocycle. In some examples, the aqueous pharmaceuticalformulations upon storage for 6 months at from about 2° C.-8° C. cancomprise at least 95% of the initial amount of the peptidomimeticmacrocycle. In some cases, the aqueous pharmaceutical formulations uponstorage for 3 months at from about 2° C.-8° C. can comprise at least 95%of the initial amount of the peptidomimetic macrocycle.

The osmolality of the aqueous pharmaceutical formulations of thedisclosure can be from about 100-600 milliosmoles per kilogram, forexample from about 220-400 milliosmoles per kilogram.

The endotoxin level of the aqueous pharmaceutical formulations of thedisclosure can be at most 2.0, 4.0, 6.0, 8.0 or 10 EU/mL². In someexamples, the endotoxin level of the aqueous pharmaceutical formulationscan be at most 4.5 EU/mL².

The aqueous pharmaceutical formulations of the disclosure can becontained in a container. The container can be a single use container ora multi-use container. In some examples, the container can be a glassvial. In some examples, the container is a pre-filled syringe to be usedalone or in an injection device. In some examples, the container is acartridge for a pen injection system, or a glass ampoule. In someexamples, the container is a 20 mL, 10 mL, or a 5 mL glass serum vial.The glass vial can comprise borosilicate glass or polycarbonate. Thecontainer can comprise stopper and/or cap. The stopper can be a rubberstopper. The container can comprise a seal for example an aluminum seal.

The aqueous pharmaceutical formulations of the disclosure can beprepared by adding the peptidomimetic macrocycle or a pharmaceuticallyacceptable salt thereof to water or an aqueous solution, wherein thepeptidomimetic macrocycle is capable of binding to the MDM2 and/or MDMXproteins. The pharmaceutically acceptable salt can be a sodium salt,potassium salt or calcium salt. In some examples, the aqueouspharmaceutical formulations can be prepared by dissolving a sodium saltof the peptidomimetic macrocycle in water. The method can furthercomprise adding a buffering agent and a stabilizing agent.

The aqueous pharmaceutical formulations of the disclosure can besuitable for administration to a subject without reconstitution ordilution. In some examples, the aqueous pharmaceutical formulations canrequire reconstitution prior to administration to a subject.Reconstitution can involve dilution with an aqueous solution, forexample with a solution of dextrose in water.

In some embodiments, the micelle forming agent in solutol-HS-15. In someembodiments, the peptidomimetic macrocycle forms a micelle in absence ofa surfactant. In some embodiments, the aqueous pharmaceuticalformulation does not form micelle.

The aqueous pharmaceutical formulations of the disclosure can furthercomprise a preservative. The preservative can be selected from a groupconsisting of benzalkonium chloride, EDTA and combination thereof.

In some examples, the preservative can be selected from a groupconsisting of phenol, meta-cresol and combination thereof.

The aqueous pharmaceutical formulations of the disclosure can furthercomprise a co-solvent. The co-solvent can be selected from a groupconsisting of dimethyl sulfoxide (DMSO), N-methylpyrrolidone (NMP),dimethylacetamide (DMA) and combinations thereof.

The molecular weight of the peptidomimetic macrocycle can be in therange of 1800-2000 D. In some examples, the peptidomimetic macrocyclehas an observed mass (m/e) in the range of 900-1000 D.

In another aspect the disclosure provides an aqueous pharmaceuticalformulation comprising a peptidomimetic macrocycle that binds to MDM2and/or MDMX proteins or a pharmaceutically acceptable salt thereof,phosphate buffering agent. D-trehalose, and polysorbate 20, wherein thepeptidomimetic macrocycle comprises an amino acid sequence which is atleast about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% identical to an aminoacid sequence in any of Table 1, Table 1a, Table 1b, and Table 1c. Insome examples, the amount of the peptidomimetic macrocycle in theaqueous pharmaceutical formulation can be equal to or greater than 15mg/mL. In some examples, the amount of D-trehalose in the aqueouspharmaceutical formulations can be about 8% w/v. The amount ofpolysorbate 20 in the aqueous pharmaceutical formulations can be about0.03% w/v. In some examples, the aqueous pharmaceutical formulationscomprise less than 2% w/v of any micelle forming agent.

The peptidomimetic macrocycle in the aqueous pharmaceutical formulationscan comprise an amino acid sequence which is at least about 60%, 65%,70%, 75%, 80%, 85%, 90%, 95% identical to an amino acid sequence in anyof Table 1, Table 1a, Table 1b, and Table 1c, and wherein thepeptidomimetic macrocycle has the formula:

wherein:each A, C, and D is independently an amino acid;each B is independently an amino acid,

[—NH-L₃-CO—], [—NH-L₃-SO₂—], or [—NH-L₃-];each E is independently an amino acid selected from the group consistingof Ala (alanine), D-Ala (D-alanine), Aib (α-aminoisobutyric acid), Sar(N-methyl glycine), and Ser (serine);

each R₁ and R₂ is independently —H, alkyl, alkenyl, alkynyl, arylalkyl,cycloalkyl, cycloalkylalkyl, heteroalkyl, or heterocycloalkyl,unsubstituted or substituted with halo, or forms a macrocycle-forminglinker U connected to the alpha position of one of said D or E aminoacids;

each R₃ independently is hydrogen, alkyl, alkenyl, alkynyl, arylalkyl,heteroalkyl, cycloalkyl, heterocycloalkyl, cycloalkylalkyl, cycloaryl,or heterocycloaryl, optionally substituted with R₅;

each L and L is independently a macrocycle-forming linker;

each L3 is independently alkylene, alkenylene, alkynylene,heteroalkylene, cycloalkylene, heterocycloalkylene, cycloarylene,hetemcycloarylene, or [—R₄—K—R₄—]_(n), each being optionally substitutedwith R₅;

each R₄ is independently alkylene, alkenylene, alkynylene,heteroalkylene, cycloalkylene, heterocycloalkylene, arylene, orheteroarylene;

each K is independently O, S, SO, SO₂, CO, CO₂, or CONR₃;

each R, is independently halogen, alkyl, —OR₆, —N(R₆)₂, —SR₆, —SOR₆,—SO₂R, —CO₂R₆, a fluorescent moiety, a radioisotope or a therapeuticagent;

each R₆ is independently —H, alkyl, alkenyl, alkynyl, arylalkyl,cycloalkylalkyl, heterocycloalkyl, a fluorescent moiety, a radioisotopeor a therapeutic agent;

each R₇ is independently —H, alkyl, alkenyl, alkynyl, arylalkyl,cycloalkyl, heteroalkyl, cycloalkylalkyl, heterocycloalkyl, cycloaryl,or heterocycloaryl, optionally substituted with R₅, or part of a cyclicstructure with a D residue;

each R₈ is independently —H, alkyl, alkenyl, alkynyl, arylalkyl,cycloalkyl, heteroalkyl, cycloalkylalkyl, heterocycloalkyl, cycloaryl,or heterocycloaryl, optionally substituted with R₅, or part of a cyclicstructure with an E residue:

each v is independently an integer from 1-1000:

each w is independently an integer from 3-1000;

u is an integer from 1-10;

each x, y and z is independently an integer from 0-10: and

each n is independently an integer from 1-5.

In some embodiments, the peptidomimetic macrocycle has formula:

wherein:

each of Xaa₃, Xaa₅, Xaa₆, Xaa₇, Xaa₈, Xaa₉, and Xaa₁₀ is individually anamino acid, wherein at least three of Xaa₃, Xaa₅, Xaa₆, Xaa₇, Xaa₈,Xaa₉, and Xaa₁₀ are the same amino acid as the amino acid at thecorresponding position of the sequencePhe-X₄-His₅-Tyr₆-Trp7-Alaa-Gln₉-Leu₁₀-X₁₁-Ser₁₂ (SEQ ID NO: 3) orPhe₃-X₄-Glu₅-Tyr₆-Trp₇-Ala₅-Gln₉-Leu₁₀/Cba₁₀-X₁₁-Ala₁₂ (SEQ ID NO: 4),where each X is an amino acid;

each D and E is independently an amino acid;

each R₁ and R₂ are independently —H, alkyl, alkenyl, alkynyl, arylalkyl,cycloalkyl, cycloalkylalkyl, heteroalkyl, or heterocycloalkyl,unsubstituted or substituted with halo-; or at least one of R₁ and R₂forms a macrocycle-forming linker L′ connected to the alpha position ofone of said D or E amino acids;

each L or L′ is independently a macrocycle-forming linker;

each R₅ is independently halogen, alkyl, —OR₆, —N(R_(b))₂, —SR₆, —SOR₆,—SO₂R₆, —CO₂R₆, a fluorescent moiety, a radioisotope or a therapeuticagent;

each R₆ is independently —H, alkyl, alkenyl, alkynyl, arylalkyl,cycloalkylalkyl, heterocycloalkyl, a fluorescent moiety, a radioisotopeor a therapeutic agent;

R₇ is —H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, heteroalkyl,cycloalkylalkyl, heterocycloalkyl, cycloaryl, or heterocycloaryl,optionally substituted with R₅, or part of a cyclic structure with a Dresidue;

R₈ is —H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, heteroalkyl,cycloalkylalkyl, heterocycloalkyl, cycloaryl, or heterocycloaryl,optionally substituted with R₅, or part of a cyclic structure with an Eresidue;

v is an integer from 1-1000; and

w is an integer from 0-1000.

In some examples, at least one of the macrocycle-forming linker in theFormulas provided herein has a formula -L₁-L₂-, wherein

L₁ and L₂ are independently alkylene, alkenylene, alkynylene,heteroalkylene, cycloalkylene, heterocycloalkylene, cycloarylene,heterocycloarylene, or [—R₄—K—R₄—]_(n), each being optionallysubstituted with R₅;each R₄ is independently alkylene, alkenylene, alkynylene,heteroalkylene, cycloalkylene, heterocycloalkylene, arylene, orheteroarylene;each K is independently O, S, SO, SO₂, CO, CO₂, or CONR₃; andeach R₃ independently is hydrogen, alkyl, alkenyl, alkynyl, arylalkyl,heteroalkyl, cycloalkyl, heterocycloalkyl, cycloalkylalkyl, cycloaryl,or heterocycloaryl, optionally substituted with R₅;each n is independently an integer from 1-5.

In some embodiments w is an integer from 3-1000, for example 3-500,3-200, 3-100, 3-50, 3-30, 3-20, or 3-10. In some examples, Xaa₅ is Gluor an amino acid analog thereof. In some examples each E isindependently an amino acid selected from Ala (alanine), D-Ala(D-alanine), Aib (α-aminoisobutyric acid), Sar (N-methyl glycine), andSer (serine). In some examples, [D]_(v) is -Leu₁-Thr₂. In some examples,w is 3-6. In some examples, w is 6-10. In some examples, w is 6. In someexamples, v is 1-10. In some examples, v is 2-10. In some examples, v is2-5. In some examples, v is 2.

In some examples, L₁ and L₂ in the Formulas above are independentlyalkylene, alkenylene, alkynylene, heteroalkylene, cycloalkylene,heterocycloalkylene, cycloarylene, or heterocycloarylene, each beingoptionally substituted with R₅. In some examples, L, and L₂ areindependently alkylene or alkenylene. In some examples, L is alkylene,alkenylene, or alkynylene. In some examples, L is alkylene. In someexamples, L is C₃-C₁₆ (alkylene. In some examples, L is C₁₀-C₁₄alkylene.

In some examples, R₁ and R₂ in the Formulas above are independently —H,alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, cycloalkylalkyl,heteroalkyl, or heterocycloalkyl, unsubstituted or substituted withhalo-. In some examples, R₁ and R₂ are H. In some examples, R₁ and R₂are independently alkyl. In some examples, R₁ and R₂ are methyl.

In some examples, x+y+z in the Formulas here is 6.

In some examples, in the Formulas here, u is 1.

In some examples, each E is Ser or Ala or an analog thereof.

In some examples, the aqueous pharmaceutical formulations comprise atleast one amino acid which is an amino acid analog.

In some examples, the peptidomimetic macrocycle in the aqueouspharmaceutical formulations is a peptidomimetic macrocycle shown inTable 1c.

In another aspect, the disclosure provides a method of treating cancerin a subject, the method comprising administering to the subject atherapeutically effective amount of an aqueous pharmaceuticalformulation of the disclosure.

In another aspect, the disclosure provides a method of treating cancerin a subject comprising administering to the subject an aqueouspharmaceutical formulation a peptidomimetic macrocycle peptidomimeticmacrocycle that is capable of binding to the MDM2 and/or MDMX proteinsand wherein the amount of the peptidomimetic macrocycle in the aqueouspharmaceutical formulation is greater than 15 mg/mL and wherein theaqueous pharmaceutical formulation contains less than 2% w/v of anymicelle forming agent. The aqueous pharmaceutical formulation canfurther comprise a buffering agent, a stabilizing agent, and/or tonicityagent.

The cancer can be selected from the group consisting of head and neckcancer, melanoma, lung cancer, breast cancer, and glioma. In someexamples, the cancer is selected from a group consisting of bladdercancer, bone cancer, breast cancer, cervical cancer, CNS cancer, coloncancer, ocular tumor, renal cancer, liver cancer, lung cancer,pancreatic cancer, choriocarcinoma (tumor of the placenta), prostatecancer, sarcoma, skin cancer, soft tissue cancer, gastric cancer, gallbladder cancer, biliary cancer, renal cancer, neoblastoma, orneuroendocrine cancer.

In another aspect, the disclosure provides a method of modulating theactivity of p53 and/or MDM2 and/or MDMX in a subject comprisingadministering to the subject an aqueous pharmaceutical formulationcomprising a peptidomimetic macrocycle capable of binding to the MDM2and/or MDMX proteins, wherein the amount of the peptidomimeticmacrocycle in the aqueous pharmaceutical formulation is greater than 15mg/mL and wherein the aqueous pharmaceutical formulation contains lessthan 2% w/v of any micelle forming agent. The aqueous pharmaceuticalformulation can further comprise a buffering agent, a tonicity agent,and/or a stabilizing agent.

In another aspect, the disclosure provides a method of antagonizing theinteraction between p53 and MDM2 and/or between p53 and MDMX proteins ina subject, the method comprising administering to the subject a aqueouspharmaceutical formulation comprising a peptidomimetic macrocyclecapable of binding to the MDM2 and/or MDMX proteins, wherein the amountof the peptidomimetic macrocycle in the aqueous pharmaceuticalformulation is greater than 15 mg/mL and wherein the aqueouspharmaceutical formulation contains less than 2% w/v of any micelleforming agent. The aqueous pharmaceutical formulation can furthercomprise a buffering agent, a stabilizing agent and/or a tonicity agent.

In another aspect, the disclosure provides a method of making an aqueouspharmaceutical formulation comprising adding greater than 15 mg/mL of apeptidomimetic macrocycle or a pharmaceutically acceptable salt thereofto water or an aqueous solution, wherein the peptidomimetic macrocycleis capable of binding to the MDM2 and/or MDMX proteins and wherein theaqueous pharmaceutical formulation comprises less than 2% w/v of anymicelle forming agent. In some examples, the method comprises adding asodium salt of the peptidomimetic macrocycle to water or an aqueoussolution. The aqueous solution can comprise a buffering agent. Theaqueous solution can also comprise a tonicity agent. The aqueoussolution can further comprise a stabilizing agent.

The method can further comprise adjusting the pH of the solutioncomprising the buffering agent and the stabilizing agent during theaddition of the peptidomimetic macrocycle. The pH can be adjusted byaddition of a pH adjusting agent. In some examples, the pH is adjustedto be in the range of from about 6.0-8.0.

The amount of the pH adjusting agent added can be from about 0.01-10%w/v, for example about 0.091% w/v. The pH adjusting agent can comprisean acid or a base. In some examples, the pH adjusting agent comprisesphosphoric acid. In some examples, the pH adjusting agent comprisessodium hydroxide, for example 0.1 N NaOH.

The method can further comprise filtration of the aqueous pharmaceuticalformulation obtained after the addition of the peptidomimetic macrocycleto the aqueous solution. The filtration is performed under vacuum orunder pressure. The filtration can comprise sterilizing filtration. Insome examples, the filtration comprises use of membrane filter. In someexamples, the membrane filter comprises cellulose or cellulosederivative, cellulosic ester (MCE), comprise polytetrafluoroethylene(PTFE), polyvinylidene, polyvinylidene chloride, or polyvinylidenefluoride. The membrane filter can have a pore size in the range fromabout 10 nm-10 μm, for example 0.2 μm. The filtration can result inclarification of the aqueous formulation. The filtering can involvepassing the aqueous pharmaceutical formulation through one or moremembrane filters.

In another aspect, the disclosure provides a kit comprising, in suitablecontainer means, an aqueous pharmaceutical formulation comprising apeptidomimetic macrocycle and instructions for administration of theaqueous pharmaceutical formulation to a human subject, wherein thepeptidomimetic macrocycle is capable of binding to MDM2 and/or MDMXproteins and wherein the amount of the peptidomimetic macrocycle in theaqueous pharmaceutical formulation is greater than 15 mg/mL and theaqueous pharmaceutical formulation comprises less than 2% w/v of anymicelle forming agent. The instructions can be for intravenousadministration of the aqueous formulation.

In some embodiments, the amount of aqueous pharmaceutical formulationmade is about 1 liter to about 100 liters. In some embodiments, theamount of aqueous pharmaceutical formulation made is about 10 litres toabout 100 litres. In some embodiments the amount of aqueouspharmaceutical formulation made is about 10 liters to about 50 liters.

A kit for formulating an aqueous pharmaceutical formulation comprising,in suitable container means, a peptidomimetic macrocycle capable ofbinding to the MDM2 and/or MDMX proteins or a pharmaceuticallyacceptable salt thereof, wherein the amount of the peptidomimeticmacrocycle in the aqueous pharmaceutical formulation is greater than 15mg/mL and the aqueous pharmaceutical formulation comprises less than 2%w/v of any micelle forming agent.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the disclosure are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present disclosure will be obtained by reference tothe following detailed description that sets forth illustrativeembodiments, in which the principles of the disclosure are utilized, andthe accompanying drawings of which:

FIG. 1 . Shows a flow diagram of the manufacturing process of anexemplary aqueous pharmaceutical formulation of the disclosure.

FIG. 2 . Shows the observation pictures for Example 5.

FIG. 3 Shows a plot of viable SJSA-1 cells (%) vs. log concentration(μM) of indicated peptide after incubation of the cells with the peptidefor 72 hr in 10% serum.

FIG. 4 Shows the 12-month stability results for Aileron peptide-1. Thedata support greater than 2 year shelf life at −20-5° C.

DETAILED DESCRIPTION OF THE DISCLOSURE

While preferred embodiments of the present disclosure have been shownand described herein, it will be obvious to those skilled in the artthat such embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the disclosure. It should beunderstood that various alternatives to the embodiments of thedisclosure described herein can be employed in practicing thedisclosure. It is intended that the following claims define the scope ofthe disclosure and that methods and structures within the scope of theseclaims and their equivalents be covered thereby.

Definitions

As used herein, the term “macrocycle” refers to a molecule having achemical structure including a ring or cycle formed by at least 9covalently bonded atoms.

As used herein, the term “peptidomimetic macrocycle” or “crosslinkedpolypeptide” refers to a compound comprising a plurality of amino acidresidues joined by a plurality of peptide bonds and at least onemacrocycle-forming linker which forms a macrocycle between a firstnaturally-occurring or non-naturally-occurring amino acid residue (oranalog) and a second naturally-occurring or non-naturally-occurringamino acid residue (or analog) within the same molecule. Peptidomimeticmacrocycle include embodiments where the macrocycle-forming linkerconnects the a carbon of the first amino acid residue (or analog) to thea carbon of the second amino acid residue (or analog). Thepeptidomimetic macrocycles optionally include one or more non-peptidebonds between one or more amino acid residues and/or amino acid analogresidues, and optionally include one or more non-naturally-occurringamino acid residues or amino acid analog residues in addition to anywhich form the macrocycle. A “corresponding uncrosslinked polypeptide”when referred to in the context of a peptidomimetic macrocycle isunderstood to relate to a polypeptide of the same length as themacrocycle and comprising the equivalent natural amino acids of thewild-type sequence corresponding to the macrocycle.

As used herein, the term “pharmaceutically acceptable salt” refers tothose salts which are suitable for pharmaceutical use, preferably foruse in humans and lower animals without undue irritation, allergicresponse and the like. Pharmaceutically acceptable salts of amines,carboxylic acids, and other types of compounds, are well known in theart. For example, S. M. Berge, et al., describe pharmaceuticallyacceptable salts in detail in J Pharmaceutical Sciences, 66: 1-19(1977), incorporated herein by reference. The salts can be prepared insitu during the final isolation and purification of the peptidomimeticmacrocycles of the invention, or separately by reacting a free base orfree acid function with a suitable reagent, as described generallybelow. For example, a free base function can be reacted with a suitableacid. Suitable pharmaceutically acceptable salts can, include metalsalts such as alkali metal salts, e. g. sodium, potassium, and lithiumsalts; and alkaline earth metal salts, e. g. calcium or magnesium salts.Examples of pharmaceutically acceptable, nontoxic acid addition saltsare salts of an amino group formed with inorganic acids such ashydrochloric acid, hydrobromic acid, hydroiodic acid, phosphoric acid,sulfuric acid and perchloric acid or with organic acids such as aceticacid, oxalic acid, maleic acid, tartaric acid, citric acid, succinicacid or malonic acid or by using other methods used in the art such asion exchange. Other pharmaceutically acceptable salts include adipate,alginate, ascorbate, aspartate, benzoate, bisulfate, borate, butyrate,camphorate, camphorsulfonate, citrate, cyclopentanepropionate,digluconate, dodecylsulfate, formate, fumarate, glucoheptonate,glycerophosphate, gluconate, hemisulfate, heptanoate. hexanoate,hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate,lauryl sulfate, malate, maleate, malonate, methanesulfonate, nicotinate,nitrate, oleate, oxalate, palmitate, pectinate, persulfate,3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate,succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate,undecanoate, valerate salts, and the like.

As used herein, the term “stability” can refer to chemical stabilityand/or physical stability. As used herein, the phrase chemical stabilitymeans the ability of a compound to maintain its chemical identity overtime. Accordingly, stability implies the ability of a chemical speciesto resist oxidation or other degradation, for example. As used herein,the phrase physical stability means the ability of a composition tomaintain consistent physical properties over time. The ability of acomposition to maintain a consistent disintegration time over time isexemplary of physical stability. In some embodiments, stability can alsorefer to the maintenance of a defined secondary structure in solution bya peptidomimetic macrocycle as measured by circular dichroism, NMR oranother biophysical measure, or resistance to proteolytic degradation invitro or in vivo. Non-limiting examples of secondary structurescontemplated herein are α-helices, 3₁₀ helices, β-turns, and s-pleatedsheets.

The term “amino acid” refers to a molecule containing both an aminogroup and a carboxyl group. Suitable amino acids include, withoutlimitation, both the D- and L-isomers of the naturally-occurring aminoacids, as well as non-naturally occurring amino acids prepared byorganic synthesis or other metabolic routes. The term amino acid, asused herein, includes, without limitation, α-amino acids, natural aminoacids, non-natural amino acids, and amino acid analogs.

The term “α-amino acid” refers to a molecule containing both an aminogroup and a carboxyl group bound to a carbon which is designated theα-carbon.

The term “β-amino acid” refers to a molecule containing both an aminogroup and a carboxyl group in a β configuration.

The term “naturally occurring amino acid” refers to any one of thetwenty amino acids commonly found in peptides synthesized in nature, andknown by the one letter abbreviations A, R, N, C, D, Q, E, G, H, I, L,K, M, F, P, S, T, W, Y and V.

The following table shows a summary of the properties of natural aminoacids:

3- 1- Side- Side-chain Letter Letter chain charge Hydropathy Amino AcidCode Code Polarity (pH 7.4) Index Alanine Ala A nonpolar neutral 1.8Arginine Arg R polar positive −4.5 Asparagine Asn N polar neutral −3.5Aspartic acid Asp D polar negative −3.5 Cysteine Cys C polar neutral 2.5Glutamic acid Glu E polar negative −3.5 Glutamine Gln Q polar neutral−3.5 Glycine Gly G nonpolar neutral −0.4 Histidine His H polar positive(10%) −3.2 neutral (90%) Isoleucine Ile I nonpolar neutral 4.5 LeucineLeu L nonpolar neutral 3.8 Lysine Lys K polar positive −3.9 MethionineMet M nonpolar neutral 1.9 Phenylalanine Phe F nonpolar neutral 2.8Proline Pro P nonpolar neutral −1.6 Serine Ser S polar neutral −0.8Threonine Thr T polar neutral −0.7 Tryptophan Trp W nonpolar neutral−0.9 Tyrosine Tyr Y polar neutral −1.3 Valine Val V nonpolar neutral 4.2

“Hydrophobic amino acids” include small hydrophobic amino acids andlarge hydrophobic amino acids. “Small hydrophobic amino acid” areglycine, alanine, proline, and analogs thereof. “Large hydrophobic aminoacids” are valine, leucine, isoleucine, phenylalanine, methionine,tryptophan, and analogs thereof. “Polar amino acids” are serine,threonine, asparagine, glutamine, cysteine, tyrosine, and analogsthereof. “Charged amino acids” are lysine, arginine, histidine,aspartate, glutamate, and analogs thereof.

The term “amino acid analog” refers to a molecule which is structurallysimilar to an amino acid and which can be substituted for an amino acidin the formation of a peptidomimetic macrocycle. Amino acid analogsinclude, without limitation, O-amino acids and amino acids where theamino or carboxy group is substituted by a similarly reactive group(e.g., substitution of the primary amine with a secondary or tertiaryamine, or substitution of the carboxy group with an ester).

The term “non-natural amino acid” refers to an amino acid which is notone of the twenty amino acids commonly found in peptides synthesized innature, and known by the one letter abbreviations A, R, N, C, D, Q, E,G, H, I, L, K, M, F, P, S, T, W, Y and V. Non-natural amino acids oramino acid analogs include, without limitation, structures according tothe following:

Amino acid analogs include β-amino acid analogs. Examples of β-aminoacid analogs include, but are not limited to, the following: cyclicβ-amino acid analogs; β-alanine; (R)-β-phenylalanine;(R)-1,2,3,4-tetrahydro-isoquinoline-3-acetic acid;(R)-3-amino-4-(1-naphthyl)-butyric acid;(R)-3-amino-4-(2,4-dichlorophenyl)butyric acid;(R)-3-amino-4-(2-chlorophenyl)-butyric acid;(R)-3-amino-4-(2-cyanophenyl)-butyric acid;(R)-3-amino-4-(2-fluorophenyl)-butyric acid;(R)-3-amino-4-(2-furyl)-butyric acid;(R)-3-amino-4-(2-methylphenyl)-butyric acid;(R)-3-amino-4-(2-naphthyl)-butyric acid;(R)-3-amino-4-(2-thienyl)-butyric acid;(R)-3-amino-4-(2-trifluoromethylphenyl)-butyric acid;(R)-3-amino-4-(3,4-dichlorophenyl)butyric acid;(R)-3-amino-4-(3,4-difluorophenyl)butyric acid;(R)-3-amino-4-(3-benzothienyl)-butyric acid;(R)-3-amino-4-(3-chlorophenyl)-butyric acid;(R)-3-amino-4-(3-cyanophenyl)-butyric acid;(R)-3-amino-4-(3-fluorophenyl)-butyric acid;(R)-3-amino-4-(3-methylphenyl)-butyric acid;(R)-3-amino-4-(3-pyridyl)-butyric acid;(R)-3-amino-4-(3-thienyl)-butyric acid;(R)-3-amino-4-(3-trifluoromethylphenyl)-butyric acid;(R)-3-amino-4-(4-bromophenyl)-butyric acid;(R)-3-amino-4-(4-chlorophenyl)-butyric acid;(R)-3-amino-4-(4-cyanophenyl)-butyric acid;(R)-3-amino-4-(4-fluorophenyl)-butyric acid;(R)-3-amino-4-(4-iodophenyl)-butyric acid;(R)-3-amino-4-(4-methylphenyl)-butyric acid;(R)-3-amino-4-(4-nitrophenyl)-butyric acid;(R)-3-amino-4-(4-pyridyl)-butyric acid;(R)-3-amino-4-(4-trifluoromethylphenyl)-butyric acid;(R)-3-amino-4-pentafluoro-phenylbutyric acid; (R)-3-amino-5-hexenoicacid; (R)-3-amino-5-hexynoic acid; (R)-3-amino-5-phenylpentanoic acid;(R)-3-amino-6-phenyl-5-hexenoic acid;(S)-1,2,3,4-tetrahydro-isoquinoline-3-acetic acid;(S)-3-amino-4-(1-naphthyl)-butyric acid;(S)-3-amino-4-(2,4-dichlorophenyl)butyric acid;(S)-3-amino-4-(2-chlorophenyl)-butyric acid;(S)-3-amino-4-(2-cyanophenyl)-butyric acid;(S)-3-amino-4-(2-fluorophenyl)-butyric acid;(S)-3-amino-4-(2-furyl)-butyric acid;(S)-3-amino-4-(2-methylphenyl)-butyric acid;(S)-3-amino-4-(2-naphthyl)-butyric acid;(S)-3-amino-4-(2-thienyl)-butyric acid;(S)-3-amino-4-(2-trifluoromethylphenyl)-butyric acid;

(S)-3-amino-4-(3,4-dichlorophenyl)butyric acid;(S)-3-amino-4-(3,4-difluorophenyl)butyric acid;(S)-3-amino-4-(3-benzothienyl)-butyric acid;(S)-3-amino-4-(3-chlorophenyl)-butyric acid;(S)-3-amino-4-(3-cyanophenyl)-butyric acid;(S)-3-amino-4-(3-fluorophenyl)-butyric acid;(S)-3-amino-4-(3-methylphenyl)-butyric acid;(S)-3-amino-4-(3-pyridyl)-butyric acid;(S)-3-amino-4-(3-thienyl)-butyric acid;(S)-3-amino-4-(3-trifluoromethylphenyl)-butyric acid;(S)-3-amino-4-(4-bromophenyl)-butyric acid;(S)-3-amino-4-(4-chlorophenyl)-butyric acid;(S)-3-amino-4-(4-cyanophenyl)-butyric acid;(S)-3-amino-4-(4-fluorophenyl)-butyric acid;(S)-3-amino-4-(4-iodophenyl)-butyric acid;(S)-3-amino-4-(4-methylphenyl)-butyric acid;(S)-3-amino-4-(4-nitrophenyl)-butyric acid;(S)-3-amino-4-(4-pyridyl)-butyric acid;(S)-3-amino-4-(4-trifluoromethylphenyl)-butyric acid;(S)-3-amino-4-pentafluoro-phenylbutyric acid; (S)-3-amino-5-hexenoicacid; (S)-3-amino-5-hexynoic acid; (S)-3-amino-5-phenylpentanoic acid;(S)-3-amino-6-phenyl-5-hexenoic acid;1,2,5,6-tetrahydropyridine-3-carboxylic acid;1,2,5,6-tetrahydropyridine-4-carboxylic acid;3-amino-3-(2-chlorophenyl)-propionic acid;3-amino-3-(2-thienyl)-propionic acid;3-amino-3-(3-bromophenyl)-propionic acid;3-amino-3-(4-chlorophenyl)-propionic acid;3-amino-3-(4-methoxyphenyl)-propionic acid;3-amino-4,4,4-trifluoro-butyric acid; 3-aminoadipic acid;D-ρ-phenylalanine; 3-leucine; L-β-homoalanine; L-β-homoaspartic acidγ-benzyl ester; L-β-homoglutamic acid δ-benzyl ester;L-β-homoisoleucine; L-β-homoleucine; L-β-homomethionine;L-β-homophenylalanine; L-β-homoproline; L-β-homotryptophan;L-β-homovaline; L-β-benzyloxycarbonyl-β-homolysine; Nω-L-β-homoarginine;O-benzyl-L-β-homohydroxyproline; O-benzyl-L-β-homoserine;O-benzyl-L-β-homothreonine; O-benzyl-L-β-homotyrosine;γ-trityl-L-β-homoasparagine; (R)-3-phenylalanine; L-β-homoaspartic acidγ-t-butyl ester; L-β-homoglutamic acid δ-t-butyl ester;L-Nω-β-homolysine; Nδ-trityl-L-β-homoglutamine;Nω-2,2,4,6,7-pentamethyl-dihydrobenzofuran-5-sulfonyl-L-β-homoarginine;O-t-butyl-L-β-homohydroxy-proline; O-t-butyl-L-β-homoserine;O-t-butyl-L-β-homothreonine; O-t-butyl-L-β-homotyrosine;2-aminocyclopentane carboxylic acid; and 2-aminocyclohexane carboxylicacid.

Amino acid analogs include analogs of alanine, valine, glycine orleucine. Examples of amino acid analogs of alanine, valine, glycine, andleucine include, but are not limited to, the following;α-methoxyglycine; α-allyl-L-alanine; α-aminoisobutyric acid;α-ethyl-leucine; β-(1-naphthyl)-D-alanine; β-(1-naphthyl)-L-alanine;β-(2-naphthyl)-D-alanine; β-(2-naphthyl)-L-alanine;β-(2-pyridyl)-D-alanine; β-(2-pyridyl)-L-alanine;β-(2-thienyl)-D-alanine; β-(2-thienyl)-L-alanine;β-(3-benzothienyl)-D-alanine; β-(3-benzothienyl)-L-alanine;β-(3-pyridyl)-D-alanine; β-(3-pyridyl)-L-alanine;β-(4-pyridyl)-D-alanine; β-(4-pyridyl)-L-alanine; β-chloro-L-alanine;β-cyano-L-alanin; β-cyclohexyl-D-alanine; β-cyclohexyl-L-alanine;β-cyclopenten-1-yl-alanine; β-cyclopentyl-alanine;β-cyclopropyl-L-Ala-OH.dicyclohexylammonium salt; β-t-butyl-D-alanine;β-t-butyl-L-alanine; γ-aminobutyric acid; L-α,β-diaminopropionic acid;2,4-dinitro-phenylglycine; 2,5-dihydro-D-phenylglycine;2-amino-4,4,4-trifluorobutyric acid; 2-fluoro-phenylglycine;3-amino-4,4,4-trifluoro-butyric acid; 3-fluoro-valine;4,4,4-trifluoro-valine; 4,5-dehydro-L-leu-OH.dicyclohexylammonium salt;4-fluoro-D-phenylglycine; 4-fluoro-L-phenylglycine;4-hydroxy-D-phenylglycine; 5,5,5-trifluoro-leucine; 6-aminohexanoicacid; cyclopentyl-D-Gly-OH.dicyclohexylammonium salt;cyclopentyl-Gly-OH.dicyclohexylammonium salt; D-α,β-diaminopropionicacid; D-α-aminobutyric acid; D-α-t-butylglycine; D-(2-thienyl)glycine;D-(3-thienyl)glycine; D-2-aminocaproic acid; D-2-indanylglycine;D-allylglycine.dicyclohexylammonium salt; D-cyclohexylglycine;D-noryaline; D-phenylglycine; β-aminobutyric acid; β-aminoisobutyricacid; (2-bromophenyl)glycine; (2-methoxyphenyl)glycine;(2-methylphenyl)glycine; (2-thiazoyl)glycine; (2-thienyl)glycine;2-amino-3-(dimethylamino)-propionic acid; L-α,β-diaminopropionic acid;L-α-aminobutyric acid; L-α-t-butylglycine; L-(3-thienyl)glycine;L-2-amino-3-(dimethylamino)-propionic acid; L-2-aminocaproic aciddicyclohexyl-ammonium salt; L-2-indanylglycine;L-allylglycine.dicyclohexyl ammonium salt; L-cyclohexylglycine;L-phenylglycine; L-propargylglycine; L-norvaline;N-α-aminomethyl-L-alanine; D-α,γ-diaminobutyric acid;L-α,γ-diaminobutyric acid; β-cyclopropyl-L-alanine;(N-β-(2,4-dinitrophenyl))-L-α,β-diaminopropionic acid;(N-γ-1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl)-D-α,β-diaminopropionicacid;(N-β-1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl)-L-α,γ-diaminopropionicacid; (N-β-4-methyltrityl)-L-α,β-diaminopropionic acid;(N-β-allyloxycarbonyl)-L-α,β-diaminopropionic acid;(N-γ-1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl)-D-α,γ-diaminobutyricacid;(N-γ-1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl)-L-α,γ-diaminobutyricacid; (N-γ-4-methyltrityl)-D-α,γ-diaminobutyric acid;(N-γ-4-methyltrityl)-L-α,γ-diaminobutyric acid;(N-γ-allyloxycarbonyl)-L-α,γ-diaminobutyric acid; D-α,γ-diaminobutyricacid; 4,5-dehydro-L-leucine; cyclopentyl-D-Gly-OH; cyclopentyl-Gly-OH;D-allylglycine; D-homocyclohexylalanine; L-1-pyrenylalanine;L-2-aminocaproic acid; L-allylglycine; L-homocyclohexylalanine; andN-(2-hydroxy-4-methoxy-Bzl)-Gly-OH.

Amino acid analogs include analogs of arginine or lysine. Examples ofamino acid analogs of arginine and lysine include, but are not limitedto, the following; citrulline; L-2-amino-3-guanidinopropionic acid;L-2-amino-3-ureidopropionic acid; L-citrulline; Lys(Mc)₂-OH; Lys(N₃)—OH;Nδ-benzyloxycarbonyl-L-omithine; Nω-nitro-D-arginine;Nω-nitro-L-arginine; α-methyl-ornithine; 2,6-diaminoheptanedioic acid;L-omithine;(Nδ-1-(4,4-dimethyl-2,6-dioxo-cyclohex-1-ylidene)ethyl)-D-ornithine;(Nδ-1-(4,4-dimethyl-2,6-dioxo-cyclohex-1-ylidene)ethyl)-L-omithine;(Nδ-4-methyltrityl)-D-ornithine; (Nδ-4-methyltrityl)-L-omithine;D-omithine; L-omithine, Arg(Me)(Pbf)-OH; Arg(Me)₂-OH (asymmetrical);Arg(Me)₂-OH (symmetrical); Lys(ivDde)-OH; Lys(Me)₂-OH.HCl; Lys(Me₃)₂-OHchloride; Nω-nitro-D-arginine; and Nω-nitro-L-arginine.

Amino acid analogs include analogs of aspartic or glutamic acids.Examples of amino acid analogs of aspartic and glutamic acids include,but are not limited to, the following; α-methyl-D-aspartic acid;α-methyl-glutamic acid; α-methyl-L-aspartic acid; γ-methylene-glutamicacid; (N-γ-ethyl)-L-glutamine; [N-α-(4-aminobenzoyl)]-L-glutamic acid;2,6-diaminopimelic acid; L-α-aminosuberic acid; D-2-aminoadipic acid;D-α-aminosuberic acid; α-aminopimelic acid; iminodiacetic acid;L-2-aminoadipic acid; threo-β-methyl-aspartic acid; γ-carboxy-D-glutamicacid γ,γ-di-t-butyl ester; γ-carboxy-L-glutamic acid γ,γ-di-t-butylester; Glu(OAll)-OH; L-Asu(OtBu)-OH; and pyroglutamic acid.

Amino acid analogs include analogs of cysteine and methionine. Examplesof amino acid analogs of cysteine and methionine include, but are notlimited to, Cys(farnesyl)-OH.Cys(famesyl)-OMe, α-methyl-methionine,Cys(2-hydroxyethyl)-OH, Cys(3-aminopropyl)-OH,2-amino-4-(ethylthio)butyric acid, buthionine, buthioninesulfoximine,ethionine, methionine methylsulfonium chloride, selenomethionine,cysteic acid, [2- (4-pyridyl)ethyl]-DL-penicillamine, [2-(4-pyridyl)ethyl]-L-cysteine, 4-methoxybenzyl-D-penicillamine,4-methoxybenzyl-L-penicillamine, 4-methylbenzyl-D-penicillamine,4-methylbenzyl-L-penicillamine, benzyl-D-cysteine, benzyl-L-cysteine,benzyl-DL-homocysteine, carbamoyl-L-cysteine, carboxyethyl-L-cysteine,carboxymethyl-L-cysteine, diphenylmethyl-L-cysteine, ethyl-L-cysteine,methyl-L-cysteine, t-butyl-D-cysteine, trityl-L-homocysteine,trityl-D-penicillamine, cystathionine, homocystine, L-homocystine,(2-aminoethyl)-L-cysteine, seleno-L-cystine, cystathionine,Cys(StBu)-OH, and acetamidomethyl-D-penicillamine.

Amino acid analogs include analogs of phenylalanine and tyrosine.Examples of amino acid analogs of phenylalanine and tyrosine includeβ-methyl-phenylalanine, β-hydroxyphenylalanine,α-methyl-3-methoxy-DL-phenylalanine, α-methyl-D-phenylalanine,α-methyl-L-phenylalanine, 1,2,3,4-tetrahydroisoquinoline-3-carboxylicacid, 2,4-dichloro-phenylalanine, 2- (trifluoromethyl)-D-phenylalanine,2-(trifluoromethyl)-L-phenylalanine, 2-bromo-D-phenylalanine,2-bromo-L-phenylalanine, 2-chloro-D-phenylalanine,2-chloro-L-phenylalanine, 2-cyano-D-phenylalanine,2-cyano-L-phenylalanine, 2-fluoro-D-phenylalanine,2-fluoro-L-phenylalanine, 2-methyl-D-phenylalanine,2-methyl-L-phenylalanine, 2-nitro-D-phenylalanine,2-nitro-L-phenylalanine, 2;4;5-trihydroxy-phenylalanine,3,4,5-trifluoro-D-phenylalanine, 3,4,5-trifluoro-L-phenylalanine,3,4-dichloro-D-phenylalanine, 3,4-dichloro-L-phenylalanine,3,4-difluoro-D-phenylalanine, 3,4-difluoro-L-phenylalanine,3,4-dihydroxy-L-phenylalanine, 3,4-dimethoxy-L-phenylalanine,3,5,3′-triiodo-L-thyronine, 3,5-diiodo-D-tyrosine,3,5-diiodo-L-tyrosine, 3,5-diiodo-L-thyronine,3-(trifluoromethyl)-D-phenylalanine,3-(trifluoromethyl)-L-phenylalanine, 3-amino-L-tyrosine,3-bromo-D-phenylalanine, 3-bromo-L-phenylalanine,3-chloro-D-phenylalanine, 3-chloro-L-phenylalanine, 3-chloro-L-tyrosine,3-cyano-D-phenylalanine, 3-cyano-L-phenylalanine,3-fluoro-D-phenylalanine, 3-fluoro-L-phenylalanine, 3-fluoro-tyrosine,3-iodo-D-phenylalanine, 3-iodo-L-phenylalanine, 3-iodo-L-tyrosine,3-methoxy-L-tyrosine, 3-methyl-D-phenylalanine,3-methyl-L-phenylalanine, 3-nitro-D-phenylalanine,3-nitro-L-phenylalanine, 3-nitro-L-tyrosine,4-(trifluoromethyl)-D-phenylalanine,4-(trifluoromethyl)-L-phenylalanine, 4-amino-D-phenylalanine,4-amino-L-phenylalanine, 4-benzoyl-D-phenylalanine,4-benzoyl-L-phenylalanine, 4-bis(2-chloroethyl)amino-L-phenylalanine,4-bromo-D-phenylalanine, 4-bromo-L-phenylalanine,4-chloro-D-phenylalanine, 4-chloro-L-phenylalanine,4-cyano-D-phenylalanine, 4-cyano-L-phenylalanine,4-fluoro-D-phenylalanine, 4-fluoro-L-phenylalanine,4-iodo-D-phenylalanine, 4-iodo-L-phenylalanine, homophenylalanine,thyroxine, 3,3-diphenylalanine, thyronine, ethyl-tyrosine, andmethyl-tyrosine.

Amino acid analogs include analogs of proline. Examples of amino acidanalogs of proline include, but are not limited to, 3,4-dehydro-proline,4-fluoro-proline, cis-4-hydroxy-proline, thiazolidine-2-carboxylic acid,and trans-4-fluoro-proline.

Amino acid analogs include analogs of serine and threonine. Examples ofamino acid analogs of serine and threonine include, but are not limitedto, 3-amino-2-hydroxy-5-methylhexanoic acid,2-amino-3-hydroxy-4-methylpentanoic acid, 2-amino-3-ethoxybutanoic acid,2-amino-3-methoxybutanoic acid, 4-amino-3-hydroxy-6-methylheptanoicacid, 2-amino-3-benzyloxypropionic acid, 2-amino-3-benzyloxypropionicacid, 2-amino-3-ethoxypropionic acid, 4-amino-3-hydroxybutanoic acid,and α-methylserine.

Amino acid analogs include analogs of tryptophan. Examples of amino acidanalogs of tryptophan include, but are not limited to the following;α-methyl-tryptophan; β-(3-benzothienyl)-D-alanine;D-(3-benzothienyl)-L-alanine; 1-methyl-tryptophan; 4-methyl-tryptophan;5-benzyloxy-tryptophan; 5-bromo-tryptophan; 5-chloro-tryptophan;5-fluoro-tryptophan; 5-hydroxy-tryptophan; 5-hydroxy-L-tryptophan;5-methoxy-tryptophan; 5-methoxy-L-tryptophan; 5-methyl-tryptophan;6-bromo-tryptophan; 6-chloro-D-tryptophan; 6-chloro-tryptophan;6-fluoro-tryptophan; 6-methyl-tryptophan; 7-benzyloxy-tryptophan;7-bromo-tryptophan; 7-methyl-tryptophan;D-1,2,3,4-tetrahydro-norharman-3-carboxylic acid;6-methoxy-1,2,3,4-tetrahydronorharman-1-carboxylic acid;7-azatryptophan; L-1,2,3,4-tetrahydro-norharman-3-carboxylic acid;5-methoxy-2-methyl-tryptophan; and 6-chloro-L-tryptophan.

In some embodiments, amino acid analogs are racemic. In someembodiments, the D isomer of the amino acid analog is used. In someembodiments, the L isomer of the amino acid analog is used. In otherembodiments, the amino acid analog comprises chiral centers that are inthe R or S configuration. In still other embodiments, the amino group(s)of a β-amino acid analog is substituted with a protecting group, e.g.,tert-butyloxycarbonyl (BOC group), 9-fluorenylmethyloxycarbonyl (FMOC),tosyl, and the like. In yet other embodiments, the carboxylic acidfunctional group of a β-amino acid analog is protected, e.g., as itsester derivative. In some embodiments the salt of the amino acid analogis used.

A “non-essential” amino acid residue is a residue that can be alteredfrom the wild-type sequence of a polypeptide without abolishing orsubstantially altering its essential biological or biochemical activity(e.g., receptor binding or activation). An “essential” amino acidresidue is a residue that, when altered from the wild-type sequence ofthe polypeptide, results in abolishing or substantially abolishing thepolypeptide's essential biological or biochemical activity.

A “conservative amino acid substitution” is one in which the amino acidresidue is replaced with an amino acid residue having a similar sidechain. Families of amino acid residues having similar side chains havebeen defined in the art. These families include amino acids with basicside chains (e.g., K, R, H), acidic side chains (e.g., D, E), unchargedpolar side chains (e.g., G, N, Q, S, T, Y, C), nonpolar side chains(e.g., A, V, L, I, P, F, M, W), beta-branched side chains (e.g., T, V,I) and aromatic side chains (e.g., Y, F, W, H). Thus, a predictednonessential amino acid residue in a polypeptide, for example, isreplaced with another amino acid residue from the same side chainfamily. Other examples of acceptable substitutions are substitutionsbased on isosteric considerations (e.g. norleucine for methionine) orother properties (e.g. 2-thienylalanine for phenylalanine, or6-Cl-tryptophan for tryptophan).

The term “capping group” refers to the chemical moiety occurring ateither the carboxy or amino terminus of the polypeptide chain of thesubject peptidomimetic macrocycle. The capping group of a carboxyterminus includes an unmodified carboxylic acid (ie —COOH) or acarboxylic acid with a substituent. For example, the carboxy terminuscan be substituted with an amino group to yield a carboxamide at theC-terminus. Various substituents include but are not limited to primaryand secondary amines, including pegylated secondary amines.Representative secondary amine capping groups for the C-terminusinclude:

The capping group of an amino terminus includes an unmodified amine (ie—NH₂) or an amine with a substituent. For example, the amino terminuscan be substituted with an acyl group to yield a carboxamide at theN-terminus. Various substituents include but are not limited tosubstituted acyl groups, including C₁-C₆ carbonyls, C₇-C₃₀ carbonyls,and pegylated carbamates. Representative capping groups for theN-terminus include, but are not limited to, 4-FBzl (4-fluoro-benzyl) andthe following:

The term “member” as used herein in conjunction with macrocycles ormacrocycle-forming linkers refers to the atoms that form or can form themacrocycle, and excludes substituent or side chain atoms. By analogy,cyclodecane, 1,2-difluoro-decane and 1,3-dimethyl cyclodecane are allconsidered ten-membered macrocycles as the hydrogen or fluorosubstituents or methyl side chains do not participate in forming themacrocycle.

The symbol “

” when used as part of a molecular structure refers to a single bond ora trans or cis double bond.

The term “amino acid side chain” refers to a moiety attached to theα-carbon (or another backbone atom) in an amino acid. For example, theamino acid side chain for alanine is methyl, the amino acid side chainfor phenylalanine is phenylmethyl, the amino acid side chain forcysteine is thiomethyl, the amino acid side chain for aspartate iscarboxymethyl, the amino acid side chain for tyrosine is4-hydroxyphenylmethyl, etc. Other non-naturally occurring amino acidside chains are also included, for example, those that occur in nature(e.g., an amino acid metabolite) or those that are made synthetically(e.g., an α,α di-substituted amino acid).

The term “α,α di-substituted amino” acid refers to a molecule or moietycontaining both an amino group and a carboxyl group bound to a carbon(the α-carbon) that is attached to two natural or non-natural amino acidside chains.

The term “polypeptide” encompasses two or more naturally ornon-naturally-occurring amino acids joined by a covalent bond (e.g., anamide bond). Polypeptides as described herein include full lengthproteins (e.g., fully processed proteins) as well as shorter amino acidsequences (e.g., fragments of naturally-occurring proteins or syntheticpolypeptide fragments).

The term “first C-terminal amino acid” refers to the amino acid which isclosest to the C-terminus. The term “second C-terminal amino acid”refers to the amino acid attached at the N-terminus of the firstC-terminal amino acid.

The term “macrocyclization reagent” or “macrocycle-forming reagent” asused herein refers to any reagent which can be used to prepare apeptidomimetic macrocycle by mediating the reaction between two reactivegroups. Reactive groups can be, for example, an azide and alkyne, inwhich case macrocyclization reagents include, without limitation, Cureagents such as reagents which provide a reactive Cu(I) species, suchas CuBr, CuI or CuOTf, as well as Cu(II) salts such as Cu(CO₂CH₃)₂,CuSO₄, and CuCl₂ that can be converted in situ to an active Cu(I)reagent by the addition of a reducing agent such as ascorbic acid orsodium ascorbate. Macrocyclization reagents can additionally include,for example, Ru reagents known in the art such as Cp*RuCl(PPh₃)₂,[Cp*RuCl]₄ or other Ru reagents which can provide a reactive Ru(II)species. In other cases, the reactive groups are terminal olefins. Insuch embodiments, the macrocyclization reagents or macrocycle-formingreagents are metathesis catalysts including, but not limited to,stabilized, late transition metal carbene complex catalysts such asGroup VIII transition metal carbene catalysts. For example, suchcatalysts are Ru and Os metal centers having a +2 oxidation state, anelectron count of 16 and pentacoordinated. In other examples, catalystshave W or Mo centers. Various catalysts are disclosed in Grubbs et al.,“Ring Closing Metathesis and Related Processes in Organic Synthesis”Acc. Chem. Res. 1995, 28, 446452, U.S. Pat. No. 5,811,515: U.S. Pat. No.7,932,397; U.S. Application No. 2011/0065915: U.S. Application No.2011/0245477: Yu et al., “Synthesis of Macrocyclic Natural Products byCatalyst-Controlled Stereoselective Ring-Closing Metathesis,” Nature2011, 479, 88: and Peryshkov et al., “Z-Selective Olefin MetathesisReactions Promoted by Tungsten Oxo Alkylidene Complexes,” J. Am. Chem.Soc. 2011, 133, 20754. In yet other cases, the reactive groups are thiolgroups. In such embodiments, the macrocyclization reagent is, forexample, a linker functionalized with two thiol-reactive groups such ashalogen groups. In some examples, the macrocyclization reagent includepalladium reagents, for example Pd(PPh₃)₄.Pd(PPh₃)₂Cl₂, Pd(dppe)Cl,Pd(dppp)Cl₂, and Pd(dppf)Cl₂. The term “halo” or “halogen” refers tofluorine, chlorine, bromine or iodine or a radical thereof.

The term “alkyl” refers to a hydrocarbon chain that is a straight chainor branched chain, containing the indicated number of carbon atoms. Forexample, C₁-C₁₀ indicates that the group has from 1 to 10 (inclusive)carbon atoms in it. In the absence of any numerical designation, “alkyl”is a chain (straight or branched) having 1 to 20 (inclusive) carbonatoms in it.

The term “alkylene” refers to a divalent alkyl (i.e., —R—).

The term “alkenyl” refers to a hydrocarbon chain that is a straightchain or branched chain having one or more carbon-carbon double bonds.The alkenyl moiety contains the indicated number of carbon atoms. Forexample, C₂-C₁₀ indicates that the group has from 2 to 10 (inclusive)carbon atoms in it. The term “lower alkenyl” refers to a C₂-C₆ alkenylchain. In the absence of any numerical designation, “alkenyl” is a chain(straight or branched) having 2 to 20 (inclusive) carbon atoms in it.

The term “alkynyl” refers to a hydrocarbon chain that is a straightchain or branched chain having one or more carbon-carbon triple bonds.The alkynyl moiety contains the indicated number of carbon atoms. Forexample, C₂-C₁₀ indicates that the group has from 2 to 10 (inclusive)carbon atoms in it. The term “lower alkynyl” refers to a C₂-C₁₀ alkynylchain. In the absence of any numerical designation, “alkynyl” is a chain(straight or branched) having 2 to 20 (inclusive) carbon atoms in it.

The term “aryl” refers to a 6-carbon monocyclic or 10-carbon bicyclicaromatic ring system wherein 0, 1, 2, 3, or 4 atoms of each ring aresubstituted by a substituent. Examples of aryl groups include phenyl,naphthyl and the like. The term “arylalkoxy” refers to an alkoxysubstituted with aryl.

“Arylalkyl” refers to an aryl group, as defined above, wherein one ofthe aryl group's hydrogen atoms has been replaced with a C₁-C₅ alkylgroup, as defined above. Representative examples of an arylalkyl groupinclude, but are not limited to, 2-methylphenyl, 3-methylphenyl,4-methylphenyl, 2-ethylphenyl, 3-ethylphenyl, 4-ethylphenyl,2-propylphenyl, 3-propylphenyl, 4-propylphenyl, 2-butylphenyl,3-butylphenyl, 4-butylphenyl, 2-pentylphenyl, 3-pentylphenyl,4-pentylphenyl, 2-isopropylphenyl, 3-isopropylphenyl, 4-isopropylphenyl,2-isobutylphenyl, 3-isobutylphenyl, 4-isobutylphenyl, 2-sec-butylphenyl,3-sec-butylphenyl, 4-sec-butylphenyl, 2-t-butylphenyl, 3-t-butylphenyland 4-t-butylphenyl.

“Arylamido” refers to an aryl group, as defined above, wherein one ofthe aryl group's hydrogen atoms has been replaced with one or more—C(O)NH₂ groups. Representative examples of an arylamido group include2-C(O)NH₂-phenyl, 3-C(O)NH₂-phenyl, 4-C(O)NH₂-phenyl, 2-C(O)NH₂-pyridyl,3-C(O)NH₂-pyridyl, and 4-C(O)NH₂-pyridyl.

“Alkylheterocycle” refers to a C₁-C₅ alkyl group, as defined above,wherein one of the C₁-C₅ alkyl group's hydrogen atoms has been replacedwith a heterocycle. Representative examples of an alkylheterocycle groupinclude, but are not limited to, —CH₂CH₂-morpholine, —CH₂CH₂-piperidine,—CH₂CH₂CH₂-morpholine, and —CH₂CH₂CH₂-imidazole.

“Alkylamido” refers to a C₁-C₅ alkyl group, as defined above, whereinone of the C₁-C₅ alkyl group's hydrogen atoms has been replaced with a—C(O)NH₂ group. Representative examples of an alkylamido group include,but are not limited to, —CH₂—C(O)NH₂, —CH₂CH₂—C(O)NH₂.—CH₂CH₂CH₂C(O)NH₂. —CH₂CH₂CH₂CH₂C(O)NH₂, —CH₂CH₂CH₂CH₂CH₂C(O)NH₂,—CH₂CH(C(O)NH₂)CH₃, —CH₂CH(C(O)NH₂)CH₂CH₃, —CH(C(O)NH₂)CH₂CH₃,—C(CH)₂CH₂C(O)NH₂, —CH₂—CH₂—NH—C(O)—CH₃, —CH₂—CH₂—NH—C(O)—CH₃—CH₃, and—CH₂—CH—NH—C(O)—CH═CH₂.

“Alkanol” refers to a C₁-C₅ alkyl group, as defined above, wherein oneof the C₁-C₅ alkyl group's hydrogen atoms has been replaced with ahydroxyl group. Representative examples of an alkanol group include, butare not limited to, —CH₂OH, —CH₂CH₂OH, —CH₂CH₂CH₂OH, —CH₂CH₂CH₂CH₂OH,—CH₂CH₂CH₂ CH₂CH₂OH, —CH₂CH(OH)CH₃, —CH₂CH(OH)CHCH₃, —CH(OH)CH₃ and—C(CH₃)₂CH₂OH.

“Alkylcarboxy” refers to a C₁-C₅ alkyl group, as defined above, whereinone of the C₁-C₅ alkyl group's hydrogen atoms has been replaced with a—COOH group. Representative examples of an alkylcarboxy group include,but are not limited to, —CH₂COOH, —CH₂CH₂COOH, —CH₂CH₂CH₂COOH,—CH₂CH₂CH₂CH₂COOH, —CH₂CH(COOH)CH₃, —CH₂CH₂CH₂CH₂CH₂COOH,—CH₂CH(COOH)CH₂CH₃, —CH(COOH)CH₂CH₃ and —C(CH₃)₂CH₂COOH.

The term “cycloalkyl” as employed herein includes saturated andpartially unsaturated cyclic hydrocarbon groups having 3 to 12 carbons,preferably 3 to 8 carbons, and more preferably 3 to 6 carbons, whereinthe cycloalkyl group additionally is optionally substituted. Somecycloalkyl groups include, without limitation, cyclopropyl, cyclobutyl,cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, andcyclooctyl.

The term “heteroaryl” refers to an aromatic 5-8 membered monocyclic,8-12 membered bicyclic, or 11-14 membered tricyclic ring system having1-3 heteroatoms if monocyclic, 1-6 heteroatoms if bicyclic, or 1-9heteroatoms if tricyclic, said heteroatoms selected from O, N, or S(e.g., carbon atoms and 1-3, 1-6, or 1-9 heteroatoms of O, N, or S ifmonocyclic, bicyclic, or tricyclic, respectively), wherein 0, 1, 2, 3,or 4 atoms of each ring are substituted by a substituent. Examples ofheteroaryl groups include pyridyl, furyl or furanyl, imidazolyl,benzimidazolyl, pyrimidinyl, thiophenyl or thienyl, quinolinyl, indolyl,thiazolyl, and the like.

The term “heteroarylalkyl” or the term “heteroaralkyl” refers to analkyl substituted with a heteroaryl. The term “heteroarylalkoxy” refersto an alkoxy substituted with heteroaryl.

The term “heteroarylalkyl” or the term “heteroaralkyl” refers to analkyl substituted with a heteroaryl. The term “heteroarylalkoxy” refersto an alkoxy substituted with heteroaryl.

The term “heterocyclyl” refers to a nonaromatic 5-8 membered monocyclic,8-12 membered bicyclic, or 11-14 membered tricyclic ring system having1-3 heteroatoms if monocyclic, 1-6 heteroatoms if bicyclic, or 1-9heteroatoms if tricyclic, said heteroatoms selected from O, N, or S(e.g., carbon atoms and 1-3, 1-6, or 1-9 heteroatoms of O, N, or S ifmonocyclic, bicyclic, or tricyclic, respectively), wherein 0, 1, 2 or 3atoms of each ring are substituted by a substituent. Examples ofheterocyclyl groups include piperazinyl, pyrrolidinyl, dioxanyl,morpholinyl, tetrahydrofuranyl, and the like.

The term “substituent” refers to a group replacing a second atom orgroup such as a hydrogen atom on any molecule, compound or moiety.Suitable substituents include, without limitation, halo, hydroxy,mercapto, oxo, nitro, haloalkyl, alkyl, alkaryl, aryl, aralkyl, alkoxy,thioalkoxy, aryloxy, amino, alkoxycarbonyl, amido, carboxy,alkanesulfonyl, alkylcarbonyl, and cyano groups.

In some embodiments, the compounds disclosed herein contain one or moreasymmetric centers and thus occur as racemates and racemic mixtures,single enantiomers, individual diastereomers and diastereomericmixtures. All such isomeric forms of these compounds are included unlessexpressly provided otherwise. In some embodiments. the compoundsdisclosed herein are also represented in multiple tautomeric forms, insuch instances, the compounds include all tautomeric forms of thecompounds described herein (e.g., if alkylation of a ring system resultsin alkylation at multiple sites, the disclosure includes all suchreaction products). All such isomeric forms of such compounds areincluded unless expressly provided otherwise. All crystal forms of thecompounds described herein are included unless expressly providedotherwise.

As used herein, the terms “increase” and “decrease” mean, respectively,to cause a statistically significantly (i.e., p<0.1) increase ordecrease of at least 5%.

As used herein, the recitation of a numerical range for a variable isintended to convey that the variable is equal to any of the valueswithin that range. Thus, for a variable which is inherently discrete,the variable is equal to any integer value within the numerical range,including the end-points of the range. Similarly, for a variable whichis inherently continuous, the variable is equal to any real value withinthe numerical range, including the end-points of the range. As anexample, and without limitation, a variable which is described as havingvalues between 0 and 2 takes the values 0, 1 or 2 if the variable isinherently discrete, and takes the values 0.0, 0.1, 0.01, 0.001, or anyother real values ≥0 and ≤2 if the variable is inherently continuous.

As used herein, unless specifically indicated otherwise, the word “or”is used in the inclusive sense of “and/or” and not the exclusive senseof “either/or.”

The term “on average” represents the mean value derived from performingat least three independent replicates for each data point.

The term “biological activity” encompasses structural and functionalproperties of a macrocycle. Biological activity is, for example,structural stability, alpha-helicity, affinity for a target, resistanceto proteolytic degradation, cell penetrability, intracellular stability,in vivo stability, or any combination thereof.

The term “binding affinity” refers to the strength of a bindinginteraction, for example between a peptidomimetic macrocycle and atarget. Binding affinity can be expressed, for example, as anequilibrium dissociation constant (“K_(D)”), which is expressed in unitswhich are a measure of concentration (e.g. M, mM, μM, nM etc).Numerically, binding affinity and K_(D) values vary inversely, such thata lower binding affinity corresponds to a higher K_(D) value, while ahigher binding affinity corresponds to a lower K_(D) value. Where highbinding affinity is desirable, “improved” binding affinity refers tohigher binding affinity and therefore lower K_(D) values.

The term “in vitro efficacy” refers to the extent to which a testcompound, such as a peptidomimetic macrocycle, produces a beneficialresult in an in vitro test system or assay. In vitro efficacy can bemeasured, for example, as an “IC₅₀” or “EC₅₀” value, which representsthe concentration of the test compound which produces 50% of the maximaleffect in the test system.

The term “ratio of in vitro efficacies” or “in vitro efficacy ratio”refers to the ratio of IC₅₀ or EC₅₀ values from a first assay (thenumerator) versus a second assay (the denominator). Consequently, animproved in vitro efficacy ratio for Assay 1 versus Assay 2 refers to alower value for the ratio expressed as IC₅₀ (Assay 1)/IC₅₀ (Assay 2) oralternatively as EC₅₀ (Assay 1)/EC₅₀ (Assay 2). This concept can also becharacterized as “improved selectivity” in Assay 1 versus Assay 2, whichcan be due either to a decrease in the IC₅₀ or EC₅₀ value for Target 1or an increase in the value for the IC₅₀ or EC₅₀ value for Target 2.

“Micelle forming agent” as used herein can be an amphiphilic compoundmeaning a compound that contains both hydrophobic groups (tails) andhydrophilic groups (heads). Micelle forming agents include surfactant,for examples ionic, non-ionic, and zwitterionic surfactants.

The details of one or more particular embodiments of the invention areset forth in the accompanying drawings and the description below. Otherfeatures, objects, and advantages of the invention will be apparent fromthe description and drawings, and from the claims.

The term “Xaa” is used in the Formulas described herein to refer to anyamino acids. This term can sometimes be followed by a number subscript,for e.g. “Xaa₆.” The number subscript in these cases may or may notrefer to the position of the amino acids “Xaa” in a sequence. Forexample in some but not all cases Xaa₆ can mean that the amino acid“Xaa” is present at the sixth position in a sequence.

OVERVIEW

In one aspect the disclosure provides aqueous pharmaceuticalformulations, for parenteral administration, comprising peptidomimeticmacrocycle or a pharmaceutically acceptable salt thereof, wherein thepeptidomimetic macrocycle binds to MDM2 and/or MDMX proteins. Theaqueous pharmaceutical formulations provided herein are aqueous solutionready for injection (for example intravenously) or aqueousconcentrations ready for dilution and injection. In some embodiments,the aqueous pharmaceutical formulations disclosed herein do not containmicelles or are essentially free of micelles. In various embodiments,the aqueous pharmaceutical formulations disclosed herein comprise lessthan 2% w/v of a micelle forming agent. In some examples the aqueouspharmaceutical formulations disclosed herein comprise less than 2%,1.9%, 1.8%, 1.7%, 1.6%, 1.5%, 1.4%, 1.3%, 1.2%, 1.1%, 1.0%, 0.9%, 08%,0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.09%, 0.08%, 0.07%, 0.06%, or0.05% w/v of a micelle forming agent. In some embodiments, the micelleforming agent is sorbitol. In some embodiments, the micelle formingagent is polyethylene glycol-poly(lactic acid). In some embodiments, themicelle forming agent is1,2-distearoyl-phosphatidylethanolamine-methyl-polyethyleneglycolconjugate. In some embodiments, no micelle forming agent is used, yetthe molecule has micelle forming properties.

The aqueous pharmaceutical formulations comprise an aqueous diluent. Insome examples, the diluent is water, purified water, water forinjection, bacteriostatic water for injection, sterile water forinjection, water for parenterals, PBS, and/or, sterile water forirrigation. In some embodiments, the diluent is water for injection. Insome embodiments, the diluent is PBS. In some embodiments, the diluentis a solution of dextrose in water, for example 5% dextrose in water.

In various embodiments, the peptidomimetic macrocycle is a cross-linkedpeptide comprising at least one macrocycle-forming linker which forms amacrocycle between a first amino acid residue (or analog) and a secondamino acid residue. In some embodiments, a peptidomimetic macrocycle hasthe Formula (I):

wherein:each A, C, and D is independently an amino acid:each B is independently an amino acid

[—NH-L₃-CO—], [—NH-L₃-SO₂—], or [—NH-L₃-];each E is independently an amino acid selected from the group consistingof Ala (alanine), D-Ala (D-alanine), Aib (α-aminoisobutyric acid), Sar(N-methyl glycine), and Ser (serine);each R₁ and R₂ is independently —H, alkyl, alkenyl, alkynyl, arylalkyl,cycloalkyl, cycloalkylalkyl, heteroalkyl, or heterocycloalkyl,unsubstituted or substituted with halo; or forms a macrocycle-forminglinker L′ connected to the alpha position of one of said D or E aminoacids;each R₃ is independently hydrogen, alkyl, alkenyl, alkynyl, arylalkyl,heteroalkyl, cycloalkyl, heterocycloalkyl, cycloalkylalkyl, cycloaryl,or heterocycloaryl, optionally substituted with R₅;each L and L′ is independently a macrocycle-forming linker;each L₃ is independently alkylene, alkenylene, alkynylene,heteroalkylene, cycloalkylene, heterocycloalkylene, cycloarylene,heterocycloarylene, or [—R₄—K—R₄—]_(n), each being optionallysubstituted with R₅;each R₄ is independently alkylene, alkenylene, alkynylene,heteroalkylene, cycloalkylene, heterocycloalkylene, arylene, orheteroarylene;each K is independently O, S, SO, SO₂, CO, CO₂, or CONR₃;each R₅ is independently halogen, alkyl, —OR₆, —N(R₆)₂, —SR₆, —SOR₆,—SO₂R₆, —CO₂R₆, a fluorescent moiety, a radioisotope or a therapeuticagent;each R₆ is independently —H, alkyl, alkenyl, alkynyl, arylalkyl,cycloalkylalkyl, heterocycloalkyl, a fluorescent moiety, a radioisotopeor a therapeutic agent:each R₇ is independently —H, alkyl, alkenyl, alkynyl, arylalkyl,cycloalkyl, heteroalkyl, cycloalkylalkyl, heterocycloalkyl, cycloaryl,or heterocycloaryl, optionally substituted with R₅, or part of a cyclicstructure with a D residue;each R₈ is independently —H, alkyl, alkenyl, alkynyl, arylalkyl,cycloalkyl, heteroalkyl, cycloalkylalkyl, heterocycloalkyl, cycloaryl,or heterocycloaryl, optionally substituted with R, or part of a cyclicstructure with an E residue;each v is independently an integer from 1-1000;each w is independently an integer from 3-1000;u is an integer from 1-10;each x, y and z is independently an integer from 0-10; and

each n is independently an integer from 1-5.

In some embodiments, the macrocycle-forming linker (L or L′) has aformula -L₁-L₂-, wherein L₁ and L₂ are independently alkylene,alkenylene, alkynylene, heteroalkylene, cycloalkylene,heterocycloalkylene, cycloarylene, heterocycloarylene, or[—R₄—K—R₄-]_(n), each being optionally substituted with R₅;

each R₄ is independently alkylene, alkenylene, alkynylene,heteroalkylene, cycloalkylene, heterocycloalkylene, arylene, orheteroarylene:each K is independently O, S, SO, SO₂, CO, CO₂, or CONR₃; andeach R₃ independently is hydrogen, alkyl, alkenyl, alkynyl, arylalkyl,heteroalkyl, cycloalkyl, heterocycloalkyl, cycloalkylalkyl, cycloaryl,or heterocycloaryl, optionally substituted with R₅;each n is an integer from 1-5.

In some embodiments the peptidomimetic macrocycle is a p53-basedpeptidomimetic macrocycle capable of binding to and modulating theactivity of p53, MDM2 and/or MDMX. In some embodiments thepeptidomimetic macrocycle is a p53-based peptidomimetic macrocycle thatinhibits the interactions between p53, MDM2 and/or MDMX proteins. Insome embodiments the peptidomimetic macrocycle is a p53-basedpeptidomimetic macrocycle that can be used for treating diseasesincluding but not limited to cancer and other hyperproliferativediseases. In some examples, the peptidomimetic macrocycle has a FormulaI and comprises an amino acid sequence which is at least about 60%, 65%,70%, 75%, 80%, 85%, 90%, 95%, 99% or more identical to an amino acidsequence in any of Table 1, Table 1a, Table 1b, and Table 1c. In someexamples, the peptidomimetic macrocycle in a peptidomimetic macrocyclefrom the any of Table 1, Table 1a, Table 1b, and Table 1c.

Any suitable dosage of peptidomimetic macrocycles can be formulated inthe aqueous pharmaceutical formulations of the present disclosure.Generally, the peptidomimetic macrocycle (or, in embodiments comprisingtwo or more peptidomimetic macrocycles, each of the peptidomimeticmacrocycle) is present in the aqueous pharmaceutical formulation in anamount greater than or equal to 1 mg/mL. For example greater than orequal to 5 mg/mL, 10 mg/mL, 15 mg/mL, 20 mg/mL, 30 mg/mL, 40 mg/mL, and50 mg/mL. In some examples, the peptidomimetic macrocycle is present inthe aqueous pharmaceutical formulation in an amount ranging from about15 mg/mL to about 100 mg/mL. In some embodiments, the peptidomimeticmacrocycle is present in the aqueous pharmaceutical formulation in anamount ranging from about 15 mg/mL to about 60 mg/mL. In someembodiments, the peptidomimetic macrocycle is present in the aqueouspharmaceutical formulation in an amount ranging from about 20 mg/mL toabout 50 mg/mL. In some embodiments, the peptidomimetic macrocycle ispresent in the aqueous pharmaceutical formulation in an amount rangingfrom about 50 mg/mL to about 100 mg/mL. In some embodiments, thepeptidomimetic macrocycles is present in the aqueous pharmaceuticalformulation in an amount ranging from about 15 mg/mL to about 20 mg/mL.In some embodiments, the peptidomimetic macrocycles is present in theaqueous pharmaceutical formulation in an amount ranging from about 15mg/mL to about 30 mg/mL. It will be readily apparent to those of skillthat the peptidomimetic macrocycle dosage can be varied depending onseveral conditions including the peptidomimetic macrocycle used, thesubject to be treated, and the disease, disorder or condition to betreated.

The aqueous pharmaceutical formulations disclosed herein canadditionally comprise a buffering agent. The buffering agent can be anyagent capable of maintaining the pH of the aqueous formulation in therange of 4.0-9.0. For example, the buffering agent is selected from agroup consisting of ammonia solution, calcium carbonate, tribasiccalcium phosphate, citric acid monohydrate, dibasic sodium phosphate,diethanolamine, malic acid, monobasic sodium phosphate,monoethanolamine, monosodium glutamate, phosphoric acid, potassiumcitrate, sodium acetate, sodium bicarbonate, sodium borate, sodiumcitrate dehydrate, sodium hydroxide, sodium lactate and triethanolamine.In some embodiments, the buffering agent can be monobasic sodiumphosphate, dibasic sodium phosphate, or a mixture thereof. The pH of theformulation can be in the range of 4.0-9.0. For example, the pH can bein the range of about 4.5-8.5, about 5.0-8.0, about 5.5-7.5, about7.0-7.5, about 7.0-8.0, about 7.0-9.0, or about 8.0-9.0. In someembodiments, the pH of the formulations is about 7.0. In someembodiments. the pH of the formulations is about 7.5. In someembodiments, the pH of the formulations is about 8.0.

The aqueous pharmaceutical formulations disclosed herein can comprise astabilizing agent. The stabilizing agent can be any pharmaceuticallyacceptable stabilizing agent. Such stabilizing agent can include, forexample antioxidants and/or surfactants. In some embodiments, thestabilizing agent is a non-ionic stabilizing agent, for example asnon-ionic surfactant. In some embodiments, the stabilizing agent is afatty acid ester. The stabilizing agent can be selected from a groupconsisting of polyoxyethylene glycol alkyl ethers, polyoxypropyleneglycol alkyl ethers, glucoside alkyl ethers, polyoxyethylene glycoloctylphenol ethers, polyoxyethylene glycol alkylphenol ethers, glycerolalkyl esters. polyoxyethylene glycol sorbitan alkyl esters, sorbitanalkyl esters, cocamide MEA, cocamide DEA. dodecyldimethylamine oxide,block copolymers of polyethylene glycol and polypropylene glycol, andpolyethoxylated tallow amine. In some examples, the stabilizing agent isa polyoxyethylene sorbitan fatty acid ester, for example polysorbate 20,polysorbate 21, polysorbate 40, polysorbate 60, polysorbate 61,polysorbate 65, polysorbate 80, polysorbate 81, polysorbate 85 orpolysorbate 120.

In some embodiments, the tonicity of the instant aqueous pharmaceuticalformulations can be adjusted, for example the tonicity of theformulations can be such that the formulations are isotonic with thephysiologic fluid. Such formulations can further comprise one or moretonicity adjusting agent (tonicity agent) to adjust the tonicity of theformulations. Any pharmaceutically acceptable tonicity agent can beused. In some examples the tonicity agents are selected from a groupconsisting of electrolytes, monosaccharides, disaccharides,polysaccharides, and water-soluble glucans. In some examples thetonicity agent in NaCl or KCL. In some examples the tonicity agent isselected from a group consisting of fructose, glucose, mannose,mannitol, sorbose, xylose, maltose, lactose, sucrose. trehalose,dextran, pullulan, dextrin, cyclodextrin, soluble starch, hydroxyethylstarch and carboxymethylcellulose. In some embodiments, the tonicityagent is trehalose.

In some examples, the formulations of the present disclosure furthercomprise one or more additional excipients. For example a preservativeor a co-solvent.

Also provided herein are methods of making the aqueous pharmaceuticalformulations disclosed herein. The method comprises adding apeptidomimetic macrocycle or a pharmaceutically acceptable salt thereofto an aqueous solution. The aqueous solution can comprise one or more ofa buffering agent, a stabilizing agent, and a tonicity agent. The methodcan further comprise adding a pH adjusting agent to maintain the pH ofthe mixture at a specified level. In some embodiments, the methodcomprises adding a desired amount of the peptidomimetic macrocycle or apharmaceutically acceptable salt (for example sodium, potassium orlithium salt) thereof to water. In some embodiments, the methodcomprises adding a desired amount of the peptidomimetic macrocycle or apharmaceutically acceptable salt thereof to an aqueous solutioncomprising a buffering agent, a stabilizing agent, and a tonicity agent.

Also provided herein is a method for treating a disease, condition ordisorder that can be treated, alleviated, or prevented by administeringto a subject an aqueous pharmaceutical formulation as described herein.The method comprises, administering to the subject the aqueouspharmaceutical formulation in an amount effective to treat, alleviate orprevent the disease, condition, or disorder. In some embodiments, thedisease, condition, or disorder is a p53 mediated disease, condition, ordisorder. In some embodiments, the disease, condition, or disorder is aMDM2 and/or MDMX mediated disease, condition, or disorder. In someembodiments, the disease, condition, or disorder is a hyperproliferativedisease and/or an inflammatory disorder. In some embodiments, thedisease, condition, or disorder is cancers and neoplastic conditions. Insome examples, the cancer is selected from a group consisting ofpancreatic cancer, bladder cancer, colon cancer, liver cancer,colorectal cancer, breast cancer, prostate cancer, renal cancer,hepatocellular cancer, lung cancer, ovarian cancer, cervical cancer,gastric cancer, esophageal cancer, head and neck cancer, melanoma,neuroendocrine cancers, CNS cancers, brain tumors, bone cancer, skincancer, ocular tumor, rectal cancer, choriocarcinoma (tumor of theplacenta), sarcoma and soft tissue cancer, testicular cancer, gallbladder cancer, and biliary cancer. In some examples, the cancer isselected from a group consisting of bladder cancer, bone cancer, breastcancer, cervical cancer, CNS cancer, colon cancer, ocular tumor, renalcancer, liver cancer, lung cancer, pancreatic cancer, choriocarcinoma(tumor of the placenta), prostate cancer, sarcoma, skin cancer, softtissue cancer, gastric cancer, gall bladder cancer, biliary cancer,renal cancer, neoblastoma, or neuroendocrine cancer. Non-limitingexamples of ocular tumor include choroidal nevus, choroidal melanoma,choroidal metastasis, choroidal hemangioma, choroidal osteoma, irismelanoma, uveal melanoma, melanocytoma, metastasis retinal capillaryhemangiomas, congenital hypertrophy of the RPE, RPE adenoma orretinoblastoma. In some cases, the cancer is selected from non-smallcell lung cancer, small-cell lung cancer, colon cancer, CNS cancer,melanoma, ovarian cancer, renal cancer, prostate cancer and breastcancer. In some examples, the cancer is breast cancer. In some examples,the cancer is gall bladder cancer. In some examples, the cancer isbiliary cancer. In some examples, the cancer is neuroendocrine cancer.In some examples, the cancer is bone cancer. In some examples, thecancer is the bone cancer is osteosarcoma. In some examples, the canceris skin cancer. In some examples, the cancer is melanoma.

In another aspect, the present disclosure provides kits for treating adisease, condition or disorder, wherein the kit comprises the aqueouspharmaceutical disclosed herein. The formulations can be packaged in anysuitable container, for example a bottle or a vial. In some examples,the formulations can be packed in glass serum vial. In some examples,the formulations can be packed in serum vials composed of borosilicateglass. In some examples, the formulations are packed in a 1 mL, a 2 mL,a 3 mL, a 4 mL, a 5 mL, a 10 mL, a 20 mL, a 30 mL, or a 50 mL glassvial. The bottles and/or vials can be equipped with stoppers and/orseals. For example, the formulations can be packaged into glass vialsequipped with Teflon stoppers and/or a flip-off cap. The flip-off capcan be a plastic cap. The glass container can be an ampoule. Theformulations can be packaged in multidose form or in single dose form.In some cases, the formulations are packaged in multidose forms. In someembodiments the formulations are packaged as single dose units. In someembodiments, the kit further comprises instructions, wherein theinstructions direct the administration of the formulation to treat thesubject in need thereof. The kit can also include a device foradministration of the formulation.

Aqueous Pharmaceutical Formulations of Peptidomimetic Macrocycles forParenteral Administration

In one aspect, the disclosure provides aqueous pharmaceuticalformulations, suitable for parenteral administration, comprisingpeptidomimetic macrocycles, as described herein and an aqueous diluent.The aqueous pharmaceutical formulations provided herein can be suitablefor intravenous, intra-arterial, intrathecal, or subcutaneousadministration. In some embodiments, the aqueous pharmaceuticalformulations are suitable for intravenous administration. The aqueouspharmaceutical formulations described herein can provide improvedsolubility and/or stability of the peptidomimetic macrocycle. Inparticular embodiments. the aqueous pharmaceutical formulations provideincreased solubility of the peptidomimetic macrocycles compared to thesolubility of the peptidomimetic macrocycles peptide in water alone.

In some examples, the aqueous diluent is water, purified water, waterfor injection, bacteriostatic water for injection, sterile water forinjection, water for parenterals, sterile water for irrigation, varioussterile solution of electrolytes and or dextrose. In some embodiments,the diluent is a pH buffered solution (e.g. phosphate-buffered saline),sterile saline solution, Ringer's solution or dextrose solution. In someembodiments, the diluent is water for injection. In some embodiments,the diluent is a solution of dextrose in water, for example 5% dextrosein water.

The aqueous pharmaceutical formulations my further comprise aco-solvent. A co-solvent is any solvent that facilitates/enhances thesolubility of the peptidomimetic macrocycles (or of the one or moreexcipients) in the aqueous diluent. The co-solvent is preferably watermiscible. In some embodiments, the co-solvent is ethyl alcohol,glycerin, polyethylene glycol, or propylene glycol. In some embodiments,the co-solvent is dimethyl sulfoxide (DMSO), N-methylpyrrolidone (NMP).dimethylacetamide (DMA) or a combination thereof.

The aqueous pharmaceutical formulations provided herein are aqueoussolution ready for injection (for example intravenously) or aqueousconcentrations ready for dilution and injection. In some embodiments,the aqueous pharmaceutical formulations disclosed herein do not containmicelles or are essentially free of micelles.

In various embodiments, the aqueous pharmaceutical formulationsdisclosed herein comprise less than 2% w/v of a micelle forming agent.In some examples the aqueous pharmaceutical formulations disclosedherein comprise less than 2%, 1.9%, 1.8%, 1.7%, 1.6%, 1.5%, 1.4%, 1.3%,1.2%, 1.1%, 1.0%, 0.9%, 08/6, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%,0.09%, 0.08%, 0.07%, 0.06%, or 0.05% w/v of a micelle forming agent. Insome examples the aqueous pharmaceutical formulations disclosed hereincomprise 0.0001%-2%, 0.0005-%2%, 0.001%-2%, 0.005%-2%, 0.01%-2%,0.05%-2%, 0.1%-2%, 0.2%-2%, 0.3%-2%, 0.4%-2%, 0.5%-2%, 0.6%-2%, 0.7%-2%,0.8%-2%, 0.9%-2%, 1.0%-2%, 1.1%-2%, 1.2%2%, 1.3%-2%, 1.4%-2%,1.5%-2%,1.6%2%, 1.7%-2%, 1.8%-2%, 1.9%-2%, 0.0001%-1.8%, 0.0005%-1.8%,0.001%-1.8%, 0.005%-1.8%, 0.01%-1.8%, 0.05%-1.8%, 1.0%-1.8%, 1.1%-1.8%,1.2%-1.8%, 1.3%-1.8%, 1.4%-1.8%, 1.5%-1.8%, 1.6%-1.8%, 0.8%-1.8%,0.9%-1.8%, 1.0%-1.8%, 1.1%-1.8%, 1.2%-%1.8%, 1.6%-1.8%, 1.6%-1.8%,0.0001%-1.6%, 0.0005%-1.6%, 0.001%-1.6%, 0.005%-1.6%, 0.01%-1.6%,0.05%-1.6%, 0.1%-1.6%, 0.2%-1.6%, 0.3%-1.6%, 0.4%-1.6%, 0.5%-1.6%,0.6%-1.6%, 0.7%-1.6%, 0.8%-1.6%, 0.9%1.6%, 1.0%-1.6%, 1.1%-1.6%,1.2%-1.6%, 1.3%-1.6%, 1.4%-1.6%, 1.5%-1.6%, 0.0001%-1.4%, 0.0005%-1.4%,0.001%-1.4%, 0.005%-1.4%, 0.01%-1.4%, 0.05%-1.4%, 0.1%-1.4%, 0.2%-1.4%,0.3%-1.4%, 0.4%-1.4%, 0.5%-1.4%, 0.6%-1.4%, 0.7%-1.4%, 0.8%- 1.4%,0.9%-1.4%, 1.0%-1.4%, 1.1%-1.4%, 1.2%-1.4%, 1.3-1.4%, 0.0001%-1.2%,0.0005%-1.2%, 0.001%-0.2%, 0.005%-1.2%, 0.01%-1.2%, 0.05%-1.2%,0.1%-1.2%, 0.2%-1.2%, 0.3%-1.2%, 0.4%-1.2%, 0.5%-1.2%, 0.6%-1.2%,0.7%-1.2%, 0.8%-1.2%, 0.9%-1.2%, 1.0%-1.2%, 1.1%-1.2%, 0.0001%-1%,0.0005%-1%, 0.001%-1%, 0.005%-1%, 0.01%-1%, 0.05%-1%, 0.1%-1%, 0.2%-1%,0.3%-1%, 0.4%-1%, 0.5%-1%, 0.6%-1%, 0.7%-1%, 0.0001%-0.8%, 0.0005%-0.8%,0.001%-0.8%, 0.005/6-0.8%, 0.01/6-0.8%, 0.05/6-0.8%, 0.1%-0.8%,0.2%-0.8%, 0.3%-0.8%, 0.4%-0.8%, 0.5%-0.8%, 0.6%-0.8%, 0.7%-0.8%,0.0001%-0.6%, 0.0005%0.6%, 0.001%-0.6%, 0.005%-0.6%, 0.01%-0.6%,0.05%-0.6%, 0.1%-0.6%, 0.2%-0.6%, 0.3%-0.6%, 0.4%-0.6%, 0.5%-0.6%,0.0001%-0.4%, 0.0005%-0.4%, 0.001%-0.4%, 0.005% Y₀-0.4%, 0.01%-0.4%,0.05% Y₀-0.4%, 0.1%-0.4%, 0.2%-0.4%, 0.3%-0.4%, 0.0001%-0.2%,0.0005%-0.2%, 0.001%-0.2%, 0.005%-0.2%, 0.01%-0.2%, 0.05%-0.2%,0.1%-0.2%, 0.0001%-0.1%, 0.0005%-0.1%, 0.001%-0.1%, 0.005%-0.1%,0.01%-0.1%, 0.05%-0.1, 0.0001%-0.05%, 0.0005%-0.05%, 0.001%-0.05/0,0.005%-0.05%, 0.01%-0.05%, 0.0001%-0.01%, 0.0005%-0.01%, 0.001%-0.01%,0.005%-0.01%, 0.0001%-0.005%, 0.0005%-0.005%, 0.001%-0.005%,0.0001%-0.001%, 0.0005%-0.001%, or 0.0001%-0.0005% w/v of a micelleforming agent. In some embodiments, the micelle forming agent issorbitol. In some embodiments, the micelle forming agent is Polyethyleneglycol-Poly(lactic acid). In some embodiments, the micelle forming agentis 1,2-Distearoyl-phosphatidylethanolamine-methyl-polyethyleneglycolconjugate. In some embodiments, no micelle-forming agent is added in theformulation, but the molecule has micelle-forming properties.

The aqueous pharmaceutical formulations disclosed herein canadditionally comprise one or more excipients suitable for aqueouspharmaceutical formulations. Exemplary excipients that can be present inthe aqueous pharmaceutical formulations described herein are describedbelow.

Buffering Agents

The aqueous pharmaceutical formulation of the disclosure can compriseone or more buffering agent, for example a pharmaceutically acceptablebuffering agent. Buffering agent can be used to control pH of theformulation and/or to maintain stability of the peptidomimeticmacrocycle. The pH range of the aqueous pharmaceutical formulation canbe pH 2 to pH 12, pH 4 to pH 9, pH 5 to pH9, or pH 6 to pH 8. In someembodiments the aqueous solution is buffered to a pH of about 5.0-9.0.In some embodiments the aqueous pharmaceutical formulation is bufferedto a pH of about 6.0-8.0. In some embodiments the pH of the aqueouspharmaceutical formulation is in the range of about 6.5-8.0, about7.0-8.0, about 7.5-8.0, about 6.0-7.5, about 6.5-7.5, about 7.0-7.5,6.0-7.0, about 6.5-7.0, about 7.0-7.5, or about 7.5-8.0. In someembodiments the aqueous solution is buffered to a pH of about 6.0, about6.5, about 7.0, about 7.5, about 8.0 or about 8.5. In some embodimentsthe aqueous pharmaceutical formulation is buffered to a pH of about 7.0,about 7.1, about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about7.7, about 7.8, about 7.9, or about 8.0. In some embodiments the aqueouspharmaceutical formulation is buffered to a pH of about 7.3-7.5.

Any buffering that can be safe for injection into mammalian tissue.particularly into humans, can be used in the pharmaceutical formulationof the disclosure. Buffering agent can be any agent capable of drivingan acidic or basic solution to a certain pH state, and then preventing achange from that state. Buffering agents that can be used in the instantaqueous pharmaceutical formulations include citrate, acetate, phosphate,maleate, tartrate, borate, carbonate, bicarbonate, succinate, orglutamate buffers.

In some examples, the buffering agent is lithium lactate, magnesiumlactate, sodium lactate, potassium lactate, calcium lactate, lithiumphosphate, sodium phosphate, potassium phosphate, calcium phosphate,sodium polyphosphate, potassium polyphosphate, sodium pyrophosphate,potassium pyrophosphate, disodium hydrogenphosphate, dipotassiumhydrogenphosphate, trisodium phosphate, tripotassium phosphate,potassium metaphosphate, lithium maleate, sodium maleate, potassiummaleate, calcium maleate, lithium tartarate, sodium tartarate, potassiumtartarate, calcium tartarate, lithium succinate, sodium succinate,potassium succinate, calcium succinate, lithium acetate, sodium acetate,potassium acetate, calcium acetate, sodium bicarbonate, potassiumbicarbonate, magnesium hydroxide, magnesium glucomate, aluminumhydroxide, aluminum hydroxide/sodium bicarbonate coprecipitate, sodiumcitrate, sodium tartarate, sodium carbonate, magnesium oxide, magnesiumhydroxide, magnesium carbonate, magnesium silicate, calciumglycerophosphate, calcium chloride, calcium hydroxide, calcium lactate,calcium carbonate, calcium bicarbonate, or mixture thereof.

In some examples, the buffering agent is a citrate buffer. Non-limitingexamples of suitable citrate buffers include lithium citratemonohydrate, sodium citrate monohydrate, potassium citrate monohydrate,calcium citrate monohydrate, lithium citrate dihydrate, sodium citratedihydrate, potassium citrate dihydrate, calcium citrate dihydrate,lithium citrate trihydrate, sodium citrate trihydrate, potassium citratetrihydrate, calcium citrate trihydrate, lithium citrate tetrahydrate,sodium citrate tetrahydrate, potassium citrate tetrahydrate, calciumcitrate tetrahydrate, lithium citrate pentahydrate, sodium citratepentahydrate, potassium citrate pentahydrate, calcium citratepentahydrate, lithium citrate hexahydrate, sodium citrate hexahydrate,potassium citrate hexahydrate, calcium citrate hexahydrate, lithiumcitrate heptahydrate, sodium citrate heptahydrate, potassium citrateheptahydrate, or calcium citrate heptahydrate.

In some examples, the buffering agent is a phosphate buffer.Non-limiting examples of suitable phosphate buffering agents that can beused in the formulations of the instant disclosure include, withoutlimitation, monobasic sodium phosphate, dibasic sodium phosphate,monobasic potassium phosphate, dibasic potassium phosphate, sodiumpyrophosphate, potassium pyrophosphate, disodium hydrogenphosphate,dipotassium hydrogenphosphate, potassium metaphosphate, calciumphosphate, tribasic, calcium phosphate, dibasic anhydrous, calciumphosphate dibasic, hydrate. In one embodiment, the buffering agent is aphosphate buffer. In one embodiment buffering agent is NaH₂PO₄. In oneembodiment, the buffering agent is Na₂HPO₄. In one embodiment thebuffering agent is a mixture of NaH₂PO₄ and Na₂HPO₄. In one embodimentbuffering agent is KH₂PO₄. In one embodiment, the buffering agent isK₂HPO₄. In one embodiment the buffering agent is a mixture of KH₂PO₄ andK₂HPO₄.

Tonicity Adjusting Agents

The aqueous pharmaceutical formulations disclosed herein can compriseone or more tonicity adjusting agents in order to adjust thetonicity/osmolarity of the formulations. For example, thetonicity/osmolarity of the aqueous pharmaceutical formulations can b^(e)adjusted to be isotonic with human plasma. This can help to avoid damageto the tissues. In various embodiments, the osmolarity of the aqueouspharmaceutical formulations disclosed herein can be in the range of 250to 1000 mOsM. For example, the osmolarity of the formulations can beabout 250-300 mOsM, 250-350 mOsM, 250-400 mOsM, 250-450 mOsM, 250-500mOsM, 250-550 mOsM, 250-600 mOsM, 250-650 mOsM, 250-700 mOsM, 250-750mOsM, 250-800 mOsM, 250-850 mOsM, 250-900 mOsM, 250-950 mOsM, 300-350mOsM, 300-400 mOsM, 300-450 mOsM, 300-500 mOsM, 300-550 mOsM, 300-600mOsM, 300-650 mOsM, 300-700 mOsM, 300-750 mOsM, 300-800 mOsM, 300-850mOsM, 300-900 mOsM, 300-950 mOsM, 300-1000 mOsM, 350-400 mOsM, 350-450mOsM, 350-500 mOsM, 350-550 mOsM, 350-600 mOsM, 350-650 mOsM, 350-700mOsM, 350-750 mOsM, 350-800 mOsM, 350-850 mOsM, 350-900 mOsM, 350-950mOsM, 350-1000 mOsM, 400-450 mOsM, 400-500 mOsM, 400-550 mOsM, 400-600mOsM, 400-650 mOsM, 400-700 mOsM, 400-750 mOsM, 400-800 mOsM, 400-850mOsM, 400-900 mOsM, 400-950 mOsM, 400-1000 mOsM, 450-500 mOsM, 450-550mOsM, 450-600 mOsM, 450-650 mOsM, 450-700 mOsM, 450-750 mOsM, 450-800mOsM, 450-850 mOsM, 450-900 mOsM, 450-950 mOsM, 450-1000 mOsM, 500-550mOsM, 500-600 mOsM, 500-650 mOsM, 500-700 mOsM, 500-750 mOsM, 500-800mOsM, 500-850 mOsM, 500-900 mOsM, 500-950 mOsM, 500-1000 mOsM, 550-600mOsM, 550-650 mOsM, 550-700 mOsM, 550-750 mOsM, 550-800 mOsM, 550-850mOsM, 550-900 mOsM, 550-950 mOsM, 550-1000 mOsM, 600-650 mOsM, 600-700mOsM, 600-750 mOsM, 600-800 mOsM, 600-850 mOsM, 600-900 mOsM, 600-950mOsM, 600-1000 mOsM, 650-700 mOsM, 650-750 mOsM, 650-800 mOsM, 650-850mOsM, 650-900 mOsM, 650-950 mOsM, 650-1000 mOsM, 700-750 mOsM, 700-800mOsM, 700-850 mOsM, 700-900 mOsM, 700-950 mOsM, 700-1000 mOsM, 750-800mOsM, 750-850 mOsM, 750-900 mOsM, 750-950 mOsM, 750-1000 mOsM, 800-850mOsM, 800-900 mOsM, 800-950 mOsM, 800-1000 mOsM, 850-900 mOsM, 850-950mOsM, 850-1000 mOsM, 900-950 mOsM, 900-1000 mOsM, or 950-1000 mOsM. Insome embodiments, the osmolarity of the formulations is in the range of250 to 450 mOsM. For example the osmolarity of the formulations can beabout 250 mOsM, about 300 mOsM, about 350 mOsM, about 400 mOsM, or about450 mOsM. In some embodiments, the formulation is isotonic with biologicfluids, i.e., the osmolarity is about 300 mOsM.

The tonicity adjusting agents can be ionic tonicity adjusting agents ornon-ionic tonicity adjusting agents.

In some embodiments, the isotonic agent is an ionic-isotonic agent. Insome embodiments, the isotonic agent is a non-ionic isotonic agent. Insome embodiments, the isotonic agent is a mixture of one or more ionicand/or non-ionic isotonic agent. In a some embodiment of the disclosurethe isotonic agent is selected from the group consisting of a salt (e.g.sodium chloride, boric acid, sodium nitrate, potassium nitrate), a sugaror sugar alcohol, an amino acid (e.g. L-glycine, L-histidine, arginine,lysine, isoleucine, aspartic acid, tryptophan, threonine), an alditol(e.g. glycerol (glycerine), 1,2-propanediol (propyleneglycol),1,3-propanediol, 1,3-butanediol, polyethyleneglycol (e.g. PEG400), ormixtures thereof. Any sugar such as mono-, di-, or polysaccharides, orwater-soluble glucans, including for example fructose, glucose, mannose,sorbose, xylose, maltose, lactose, sucrose, trehalose, dextran,pullulan, dextrin, cyclodextrin, soluble starch, hydroxyethyl starch andcarboxymethylcellulose-Na can be used. In some examples, the tonicityadjusting agent is selected from a group consisting of dextrose,glycerin, mannitol, trehalose, potassium chloride and sodium chloride.In some example, the tonicity adjusting agent is trehalose, for exampleD- trehalose. In some example, the tonicity adjusting agent is sodiumchloride. In some example, the tonicity adjusting agent is potassiumchloride. The use of an tonicity adjusting agent in aqueouspharmaceutical formulations is well-known to the skilled person. Forconvenience reference is made to Remington: The Science and Practice ofPharmacy, 19^(th) edition, 1995.

Stabilizing Agent

The aqueous pharmaceutical formulations described herein comprise astabilizing agent. Non-limiting examples of stabilizing agents that canbe used include acacia, agar, albumin, alginic acid, aluminum stearate,ammonium alginate, arabinose, arginine HCL, ascorbic acid, ascorbylpalmitate, bentonite, butylated hydroxytoluene, calcium alginate,calcium stearate, carboxymethylcellulose calcium. carboxymethylcellulosesodium, carrageenan, cellobiose, cellulose ceratonia, colloidal silicondioxide, cyclodextrins, diethanolamine, dextran, edentates,ethylcellulose, ethylene glycol palmitostearate, fructose, gentiobiose,glucose, glucosamine, glycine, glycerin monostearate, hydroxypropylcellulose, hydroxyethyl starch, hypromellose, hyaluronic acid, invertsugar, isomaltose, lactose, lecithin, magnesium aluminum silicate,mannose, mannitol, maltose, mineral oil and lanolin alcohols,monoethanolamine, N-methyl pyrollidone, pectin, polacrilin potassium,poloxamer (for example poloxamer 124, poloxamer 188, poloxamer 237,poloxamer 338, or poloxamer 407), polyoxyethylene sobitan fatty acidesters, polyvinyl alcohol, potassium alginate, potassium chloride,povidone (for example povidone K-12, povidone K-15, povidone K-17,povidone K-25, povidone K-20, povidone K-60, povidone K-90, or povidoneK-120), propyl gallate, propylene glycol, propylene glycol alginate,raffinose, sodium acetate, sodium alginate, sodium borate, sodiumchloride, sodium stearyl fumarate, sorbitol, stearyl alcohol, sucrose,sulfobutylether β-cyclodextrin, starch, trehalose, white wax, xanthangum, xylitol, yellow wax and zinc acetate.

In some embodiments, the stabilizing agent is a polyoxyethylene sobitanfatty acid ester, for example polysorbate 20, polysorbate 21,polysorbate 40, polysorbate 60, polysorbate 61, polysorbate 65,polysorbate 80, polysorbate 81, polysorbate 85 or polysorbate 120. Insome embodiments, the stabilizing agent is polysorbate 20.

In some embodiments, the stabilizing agent is polysorbate 21. In someembodiments, the stabilizing agent is polysorbate 40. In someembodiments, the stabilizing agent is polysorbate 60. In someembodiments, the stabilizing agent is polysorbate 61. In someembodiments. the stabilizing agent is polysorbate 65. In someembodiments, the stabilizing agent is polysorbate 80. In someembodiments, the stabilizing agent is polysorbate 81. In someembodiments, the stabilizing agent is polysorbate 85. In someembodiments, the stabilizing agent is polysorbate 120.

Preservatives-Antioxidants, Antimicrobial and Chelating Agents

The aqueous pharmaceutical formulations disclosed herein can compriseone or more antioxidants in order to prevent/minimize the oxidation ofthe peptidomimetic macrocycle and/or the excipients present in theformulation The antioxidants can also be used as a stabilizing agent.The anti-oxidants which can be used to form aqueous pharmaceuticalformulations the disclosure include, but are not limited to, propyl,octyl and dodecyl esters of gallic acid, butylated hydroxyanisole (BHA,usually purchased as a mixture of ortho and meta isomers), green teaextract, uric acid, cysteine, pyruvate, nordihydroguaiaretic acid,ascorbic acid, salts of ascorbic acid such as ascorbyl palmitate andsodium ascorbate, ascorbyl glucosamine, vitamin E (i.e., tocopherolssuch as α-tocopherol), derivatives of vitamin E (e.g., tocopherylacetate), retinoids such as retinoic acid, retinol, trans-retinol,cis-retinol, mixtures of trans-retinol and cis-retinol, 3-dehydroretinoland derivatives of vitamin A (e.g., retinyl acetate, retinal and retinylpalmitate, also known as tetinyl palmitate), sodium citrate, sodiumsulfite, sodium thiosulfate, sodium bisulfate, lycopene, anthocyanids,bioflavinoids (e.g., hesperitin, naringen, rutin and quercetin),superoxide dismutase, glutathione peroxidase, butylated hydroxytoluene(BHT), indole-3-carbinol, pycnogenol, melatonin, sulforaphane,pregnenolone, lipoic acid and 4-hydroxy-5-methyl-3[2H]-furanone. Invarious embodiments, one or more of the above antioxidants are excluded,or are present in less than effective amounts.

In some embodiments the antioxidant is ascorbic acid, citric acid,acetylcysteine, sulfurous acid salts (such as bisulfite, metasulfite),and monothioglyercol.

The aqueous pharmaceutical formulations can comprise one or moreantimicrobial agent. Suitable antimicrobial agents that can be usedinclude alcohol, benzalkonium chloride, benzyl alcohol, boric acid,bronopol, butylated hydroxyanisole, butylparaben, carbon dioxide,bentonite, cetrimide, cetylpyridinium chloride, chlorbutanol,chlorhexidine, chlorobutanol, chlorocresol, chloroxylenol, cresol (metacresol), dimethyl ether, ethylparaben, glycerin, hexetidine, imidurea,inactivation by magnesium trisilicate, isopropyl alcohol, lactic acid,methylparaben, monothioglycerol, parabens (methyl, propyl, butyl),phenol. phenoxyethanol, phenylethyl alcohol, phenylmercuric salts(acetate, borate, nitrate) phenylmercuric borate, phenylmercuricnitrate, potassium benzoate, potassium metabisulfite, potassium sorbate,propionic acid, propyl gallate, propylene glycol, propylparaben, sodiumacetate, sodium benzoate, sodium borate, sodium lactate, sodiummetabisulfite, sodium propionate, sodium sulfite, sorbic acid,synergists, edetic acid, thimerosal, xylitol, or other agents known tothose skilled in the art. In some embodiments, the antimicrobial agentused is methyl paraben, ethyl paraben, propyl paraben, or a combinationthereof. In some embodiments, the antimicrobial agent used isbenzalkonium chloride.

The aqueous pharmaceutical formulations disclosed herein can compriseone or more chelating agents.

Non-limiting examples of chelating agents which can be used to formaqueous pharmaceutical formulations of the disclosure include, but arenot limited to, ethylene diaminetetraacetic acid (EDTA), EDTA disodium,calcium disodium edetate, EDTA trisodium, albumin, transferrin,desferoxamine, desferal, desferoxamine mesylate, EDTA tetrasodium andEDTA dipotassium, sodium metasilicate, citric acid monohydrate, fumaricacid, malic acid, maltol, or combinations of any of these. In someembodiments, the formulations of the current disclosure contain no oressentially no chelating agents. In some further embodiments, theformulations are solutions containing no chelating agents.

In some embodiments, the aqueous pharmaceutical formulations of thedisclosure comprise no or essentially no preservatives. In some furtherembodiments, the aqueous pharmaceutical formulations are solutionscontaining no preservatives.

Surfactants

The solubility of the components of the present formulations can beenhanced by a surfactant or other appropriate co-solvent in thecomposition. Such co-solvents include polysorbate 20, 60, and 80,Pluronic® F68, F-84 and P-103, cyclodextrin, or other agents known tothose skilled in the art. Such co-solvents can be employed at a level offrom about 0.01% to 2% by weight. In addition, the surfactant can beused to prevent aggregation of the compound.

Surfactants which can be used to form aqueous pharmaceuticalformulations include, but are not limited to, hydrophilic surfactants,lipophilic surfactants, and mixtures thereof. That is, a mixture ofhydrophilic surfactants can be employed, a mixture of lipophilicsurfactants can be employed, or a mixture of at least one hydrophilicsurfactant and at least one lipophilic surfactant can be employed.

In some embodiments of the disclosure, the surfactant can be the sodiumsalt form of the compound, which can include the monosodium salt form.Suitable sodium salt surfactants can be selected based on desirableproperties, including high speed of polymerization, small resultantparticle sizes suitable for delivery, good polymerization yields,stability including freeze-thaw and shelf-life stability, improvedsurface tension properties, and lubrication properties.

The surfactant can be any suitable, non-toxic compound that isnon-reactive with the medicament and that substantially reduces thesurface tension between the medicament, the excipient and the site ofadministration. Some useful surfactants are: oleic acid available underthe trade names Mednique 6322 and Emersol 6321 (from Cognis Corp.,Cincinnati, Ohio); cetylpyridinium chloride (from Arrow Chemical, Inc.Westwood, N.J.): soya lecithin available under the trade name Epikuron200 (from Lucas Meyer Decatur, Ill.); polyoxyethylene(20) sorbitanmonolaurate available under the tradename Tween 20 (from ICI SpecialtyChemicals, Wilmington, Del.): polyoxyethylene(20) sorbitan monostearateavailable under the tradename Tween 60 (from ICI); polyoxyethylene(20)sorbitan monooleate available under the tradename Tween 80 (from ICI);polyoxyethylene (10) stearyl ether available under the tradename Brij 76(from ICI); polyoxyethylene (2) oleyl ether available under thetradename Brij 92 (frown ICI);Polyoxyethylene-polyoxypropylene-ethylenediamine block copolymeravailable under the tradename Tetronic 150 R1 (from BASF);polyoxypropylene-polyoxyethylene block copolymers available under thetrade names Pluronic L-92, Pluronic L-121 end Pluronic F 68 (from BASF);castor oil ethoxylate available under the tradename Alkasurf CO-40 (fromRhone-Poulenc Mississauga Ontario, Canada): and mixtures thereof.

A suitable hydrophilic surfactant can generally have an HLB value of atleast 10, while suitable lipophilic surfactants can generally have anHLB value of or less than about 10. An empirical parameter used tocharacterize the relative hydrophilicity and hydrophobicity of non-ionicamphiphilic compounds is the hydrophilic-lipophilic balance (“HLB”value). Surfactants with lower HLB values are more lipophilic orhydrophobic, and have greater solubility in oils, while surfactants withhigher HLB values are more hydrophilic, and have greater solubility inaqueous solutions. Hydrophilic surfactants are generally considered tobe those compounds having an HLB value greater than about 10, as well asanionic, cationic, or zwitterionic compounds for which the HLB scale isnot generally applicable. Similarly, lipophilic (i.e., hydrophobic)surfactants are compounds having an HLB value equal to or less thanabout 10. However, HLB value of a surfactant is merely a rough guidegenerally used to enable formulation of industrial, pharmaceutical andcosmetic emulsions.

Hydrophilic surfactants can be either ionic or non-ionic. Suitable ionicsurfactants include, but are not limited to, alkylammonium salts:fusidic acid salts; fatty acid derivatives of amino acids,oligopeptides, and polypeptides: glyceride derivatives of amino acids,oligopeptides, and polypeptides; lecithins and hydrogenated lecithins;lysolecithins and hydrogenated lysolecithins; phospholipids andderivatives thereof; lysophospholipids and derivatives thereof:carnitine fatty acid ester salts; salts of alkylsulfates; fatty acidsalts; sodium docusate: acyl lactylates: mono- and di-acetylatedtartaric acid esters of mono- and di-glycerides: succinylated mono- anddi-glycerides; citric acid esters of mono- and di-glycerides; andmixtures thereof.

Within the aforementioned group, some ionic surfactants include, by wayof example: lecithins, lysolecithin, phospholipids, lysophospholipidsand derivatives thereof; carnitine fatty acid ester salts: salts ofalkylsulfates; fatty acid salts; sodium docusate: acyl lactylates; mono-and di-acetylated tartaric acid esters of mono- and di-glycerides;succinylated mono- and di-glycerides; citric acid esters of mono- anddi-glycerides; and mixtures thereof.

Ionic surfactants can be the ionized forms of lecithin, lysolecithin,phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol,phosphatidic acid, phosphatidylserine, lysophosphatidylcholine,lysophosphatidylethanolamine, lysophosphatidylglycerol, lysophosphatidicacid, lysophosphatidylserine, PEG-phosphatidylethanolamine,PVP-phosphatidylethanolamine, lactylic esters of fatty acids,stearoyl-2-lactylate, stearoyl lactylate, succinylated monoglycerides,mono/diacetylated tartaric acid esters of mono/diglycerides, citric acidesters of mono/diglycerides. cholylsarcosine, caproate, caprylate,caprate, laurate, myristate, palmitate, oleate, ricinoleate, linoleate,linolenate, stearate, lauryl sulfate, teracecyl sulfate, docusate,lauroyl carnitines, palmitoyl carnitines, myristoyl camitines, and saltsand mixtures thereof.

Hydrophilic non-ionic surfactants can include, but not limited to,alkylglucosides; alkylmaltosides; alkylthioglucosides: laurylmacrogolglycerides: polyoxyalkylene alkyl ethers such as polyethyleneglycol alkyl ethers: polyoxyalkylene alkylphenols such as polyethyleneglycol alkyl phenols: polyoxyalkylene alkyl phenol fatty acid esterssuch as polyethylene glycol fatty acids monoesters and polyethyleneglycol fatty acids diesters; polyethylene glycol glycerol fatty acidesters; polyglycerol fatty acid esters; polyoxyalkylene sorbitan fattyacid esters such as polyethylene glycol sorbitan fatty acid esters;hydrophilic transesterification products of a polyol with at least onemember of the group consisting of glycerides, vegetable oils,hydrogenated vegetable oils, fatty acids, and sterols: polyoxyethylenesterols, derivatives, and analogues thereof; polyoxyethylated vitaminsand derivatives thereof: polyoxyethylene-polyoxypropylene blockcopolymers; and mixtures thereof; polyethylene glycol sorbitan fattyacid esters and hydrophilic transesterification products of a polyolwith at least one member of the group consisting of triglycerides,vegetable oils, and hydrogenated vegetable oils. The polyol can beglycerol, ethylene glycol, polyethylene glycol, sorbitol, propyleneglycol, pentaerythritol, or a saccharide.

Other hydrophilic-non-ionic surfactants include, without limitation,PEG-10 laurate, PEG-12 laurate, PEG-20 laurate, PEG-32 laurate, PEG-32dilaurate, PEG-12 oleate, PEG-15 oleate, PEG-20 oleate, PEG-20 dioleate,PEG-32 oleate, PEG-200 oleate, PEG-400 oleate, PEG-15 stearate, PEG-32distearate, PEG-40 stearate.

PEG-100 stearate, PEG-20 dilaurate, PEG-25 glyceryl trioleate, PEG-32dioleate, PEG-20 glyceryl laurate, PEG-30 glyceryl laurate, PEG-20glyceryl stearate, PEG-20 glyceryl oleate, PEG-30 glyceryl oleate,PEG-30 glyceryl laurate, PEG-40 glyceryl laurate, PEG-40 palm kerneloil, PEG-50 hydrogenated castor oil, PEG-40 castor oil, PEG-35 castoroil, PEG-60 castor oil, PEG-40 hydrogenated castor oil, PEG-60hydrogenated castor oil, PEG-60 corn oil, PEG-6 caprate/caprylateglycerides, PEG-8 caprate/caprylate glycerides, polyglyceryl-10 laurate,PEG-30 cholesterol, PEG-25 phyto sterol. PEG-30 soya sterol, PEG-20trioleate, PEG-40 sorbitan oleate, PEG-80 sorbitan laurate, polysorbate20, polysorbate 80, POE-9 lauryl ether, POE-23 lauryl ether, POE-10oleyl ether, POE-20 oleyl ether, POE-20 stearyl ether, tocopherylPEG-100 succinate, PEG-24 cholesterol, polyglyceryl-10 oleate, Tween 40,Tween 60, sucrose monostearate, sucrose monolaurate, sucrosemonopalmitate, PEG 10-100 nonyl phenol series, PEG 15-100 octyl phenolseries, and poloxamers.

Suitable lipophilic surfactants include, by way of example only: fattyalcohols: glycerol fatty acid esters; acetylated glycerol fatty acidesters; lower alcohol fatty acids esters; propylene glycol fatty acidesters; sorbitan fatty acid esters; polyethylene glycol sorbitan fattyacid esters; sterols and sterol derivatives; polyoxyethylated sterolsand sterol derivatives; polyethylene glycol alkyl ethers: sugar esters;sugar ethers; lactic acid derivatives of mono- and di-glycerides:hydrophobic transesterification products of a polyol with at least onemember of the group consisting of glycerides, vegetable oils,hydrogenated vegetable oils, fatty acids and sterols; oil-solublevitamins/vitamin derivatives; and mixtures thereof. Within this group,some lipophilic surfactants include glycerol fatty acid esters,propylene glycol fatty acid esters, and mixtures thereof, or arehydrophobic transesterification products of a polyol with at least onemember of the group consisting of vegetable oils, hydrogenated vegetableoils, and triglycerides.

In some embodiments the formulations of the disclosure contain nosurfactants. In some embodiments, the formulations of the disclosure areintravenous formulations containing no surfactants. In some furtherembodiments the formulations contain substantially no surfactant, i.e.contain less than approximately 0.0001% by weight of surfactants. Insome embodiments, the formulations contain essentially no surfactants.

If desired, however, the formulations can contain surface-active agentsconventionally employed, such as oleic acid, lecithin, sorbitantrioleate, cetylpyridinium chloride, benzalkonium chloride,polyoxyethylene (20) sorbitan monolaurate, polyoxyethylene (20) sorbitanmonostearate, polyoxyethylene (20) sorbitan mono-oleate,polyoxypropylene/polyoxyethylene block copolymers,polyoxypropylene/polyoxyethylene/ethylene diamine block copolymers,ethoxylated castor oil and the like, where the proportion ofsurface-active agents, if present, can be about 0.0001 to 1% by weight,or about 0.001 to 0.1% by weight, based on the total formulation. Othersuitable surfactant/emulsifying agents would be known to one of skill inthe art and are listed in the CTFA International Cosmetic IngredientDictionary and Handbook, Vol. 2, 7th Edition (1997).

The aqueous pharmaceutical formulations of the disclosure can furtherinclude other pharmacological active ingredients as far as they do notcontradict the purpose of the present disclosure. The aqueouspharmaceutical formulations for example can comprise solubilizingagents, bulking agents, dissolution enhancers, wetting agents,emulsifiers, suspending agents, antibacterial agents, sweeteners,perfuming agents, flavoring agents, and combinations thereof.

Some of the excipients or additives can have more than one possiblefunction or use, depending on their properties and the nature of theformulation. In a combination of plural active ingredients, theirrespective contents can be suitably increased or decreased inconsideration of their effects and safety.

Peptidomimetic Macrocycles

In some embodiments, a peptidomimetic macrocycle has the Formula (I):

wherein:each A, C, and D is independently an amino acid;each B is independently an amino acid

[—NH-L-CO—], [—NH-L₃-SO₂—], or [—NH-L₃-];

each E is independently an amino acid selected from the group consistingof Ala (alanine), D-Ala (D-alanine), Aib (α-aminoisobutyric acid), Sar(N-methyl glycine), and Ser (serine);

each R, independently is hydrogen. alkyl, alkenyl, alkynyl, arylalkyl,heteroalkyl, cycloalkyl, heterocycloalkyl, cycloalkylalkyl, cycloaryl,or heterocycloaryl, optionally substituted with R₅;

each R₁ and R₂ is independently —H, alkyl, alkenyl, alkynyl, arylalkyl,cycloalkyl, cycloalkylalkyl, heteroalkyl, or heterocycloalkyl,unsubstituted or substituted with halo-: or forms a macrocycle-forminglinker L′ connected to the alpha position of one of said D or E aminoacids;

each L and L′ is independently a macrocycle-forming linker:

each L₃ is independently alkylene, alkenylene, alkynylene,heteroalkylene, cycloalkylene, heterocycloalkylene, cycloarylene,heterocycloarylene, or [—R₄—K—R₄—], each being optionally substitutedwith R₅;each R₄ is independently alkylene, alkenylene, alkynylene,heteroalkylene, cycloalkylene, heterocycloalkylene, arylene, orheteroarylene;each K is independently O, S, SO, SO₂, CO, CO₂, or CONR₃;

each R₅ is independently halogen, alkyl, —OR, —N(R₆)₂, —SR, —SOR₆,—SO₂R₆, —CO₂R₆, a fluorescent moiety, a radioisotope or a therapeuticagent;

each R₆ is independently —H, alkyl, alkenyl, alkynyl, arylalkyl,cycloalkylalkyl, heterocycloalkyl, a fluorescent moiety, a radioisotopeor a therapeutic agent;

each R₇ is independently —H, alkyl, alkenyl, alkynyl, arylalkyl,cycloalkyl, heteroalkyl, cycloalkylalkyl, heterocycloalkyl, cycloaryl,or heterocycloaryl, optionally substituted with R₅, or part of a cyclicstructure with a D residue;

each R₈ is independently —H, alkyl, alkenyl, alkynyl, arylalkyl,cycloalkyl, heteroalkyl, cycloalkylalkyl, heterocycloalkyl, cycloaryl,or heterocycloaryl, optionally substituted with R₅, or part of a cyclicstructure with an E residue;

each v is independently an integer;

each w is independently an integer from 3-1000;

u is an integer from 1-10;

each x, y and z is independently an integer from 0-10; and

each n is independently an integer from 1-5.

In some embodiments, each v and w is independently integers between1-30. In some embodiments, w is an integer from 3-1000, for example3-500, 3-200, 3-100, 3-50, 3-30, 3-20, or 3-10. In some embodiments, thesum of x+y+z is 3 or 6. In some embodiments, the sum of x+y+z is 3. Inother embodiments, the sum of x+y+z is 6.

In some embodiments, peptidomimetic macrocycles are also provided of theformula:

wherein:

each of Xaa₃, Xaa₅, Xaa₆, Xaa₇, Xaa₈, Xaa₉, and Xaa₁₀ is individually anamino acid, wherein at least three of Xaa₃, Xaa₅, Xaa₆, Xaa₇, Xaa₈,Xaa₄, and Xaa₁₀ are the same amino acid as the amino acid at thecorresponding position of the sequencePhe₃-X₄-His₅-Tyr₆-Trp₇-Ala₈-Gln₉-Leu₁₀-X₁₁-Ser₁₂ (SEQ ID NO: 3), whereeach X is an amino acid;

each D and E is independently an amino acid;

each R₁ and R₂ is independently —H, alkyl, alkenyl, alkynyl, arylalkyl,cycloalkyl, cycloalkylalkyl, heteroalkyl, or heterocycloalkyl,unsubstituted or substituted with halo-; or at least one of R₁ and R₂forms a macrocycle-forming linker L′ connected to the alpha position ofone of said D or E amino acids;

each L or L′ is independently a macrocycle-forming linker;

each R₅ is independently halogen, alkyl, —OR₆, —N(R₆)₂, —SR₆, —SOR₆,—SO₂R₆, —CO₂R₆, a fluorescent moiety, a radioisotope or a therapeuticagent;

each R₆ is independently —H, alkyl, alkenyl, alkynyl, arylalkyl,cycloalkylalkyl, heterocycloalkyl, a fluorescent moiety, a radioisotopeor a therapeutic agent;

each R₇ is independently —H, alkyl, alkenyl, alkynyl, arylalkyl,cycloalkyl, heteroalkyl, cycloalkylalkyl, heterocycloalkyl, cycloaryl,or heterocycloaryl, optionally substituted with R₅, or part of a cyclicstructure with a D residue:

each R₈ is independently —H, alkyl, alkenyl, alkynyl, arylalkyl,cycloalkyl, heteroalkyl, cycloalkylalkyl, heterocycloalkyl, cycloaryl,or heterocycloaryl, optionally substituted with R₅, or part of a cyclicstructure with an E residue;

v is an integer from 1-1000, for example 1-500, 1-200, 1-100, 1-50,1-30, 1-20 or 1-10; and

w is an integer from 3-1000, for example 3-500, 3-200, 3-100, 3-50,3-30, 3-20, or 3-10.

In some embodiments, each v and w is independently an integer between1-30. In some embodiments, w is an integer from 3-1000, for example3-500, 3-200, 3-100, 3-50, 3-30, 3-20, or 3-10. In some embodiments, thesum of x+y+z is 3 or 6. In some embodiments, the sum of x+y+z is 3. Inother embodiments, the sum of x+y+z is 6.

In some embodiments of any of the Formulas described herein, at leastthree of Xaa₃, Xaa₅, Xaa₆, Xaa₇, Xaa₈, Xaa₉, and Xaa₁₀ are the sameamino acid as the amino acid at the corresponding position of thesequence Phe₃-X₄-His₅-Tyr₆-Trp₇-Ala₈-Gln₉-Leu₁₀-X₁₁-Ser₁₂ (SEQ ID NO:3). In other embodiments, at least four of Xaa₃, Xaa₅, Xaa₆, Xaa₇, Xaa₈,Xaa₉, and Xaa₁₀ are the same amino acid as the amino acid at thecorresponding position of the sequencePhe₃-X₄-His₅-Tyr₆-Trp₇-Ala₈-Gln₉-Leu₁₀-X₁₁-Ser₁₂ (SEQ ID NO: 3). Inother embodiments, at least five of Xaa₃, Xaa₅, Xaa₆, Xaa₇, Xaa₈, Xaa₉,and Xaa₁₀ are the same amino acid as the amino acid at the correspondingposition of the sequencePhe₃-X₄-His₅-Tyr₆-Trp₇-Ala₈-Gln₉-Leu₁₀-X₁₁-Ser₁₂ (SEQ ID NO: 3). Inother embodiments, at least six of Xaa₃, Xaa₅, Xaa₆, Xaa₇, Xaa₈, Xaa₉,and Xaa₁₀ are the same amino acid as the amino acid at the correspondingposition of the sequencePhe₃-X₄-His₅-Tyr₆-Trp₇-Ala₈-Gln₉-Leu₁₀-X₁₁-Ser₁₂ (SEQ ID NO: 3). Inother embodiments, at least seven of Xaa₃, Xaa₅, Xaa₆, Xaa₇, Xaa₈, Xaa₉,and Xaa₁₀ are the same amino acid as the amino acid at the correspondingposition of the sequencePhe₃-X₄-His₅-Tyr₆-Trp₇-Ala₈-Gln₉-Leu₁₀-X₁₁-Ser₁₂ (SEQ ID NO: 3).

In some embodiments, a peptidomimetic macrocycle has the Formula:

wherein:

each of Xaa₃, Xaa₅, Xaa₆, Xaa₇, Xaa₈, Xaa₉, and Xaa₁₀ is individually anamino acid, wherein at least three of Xaa₃, Xaa₅, Xaa₆, Xaa₇, Xaa₈,Xaa₉, and Xaa₁₀ are the same amino acid as the amino acid at thecorresponding position of the sequencePhe₃-X₄-Glu₅-Tyr₆-Trp₇-Ala₈-Gln₉-Leu₁₀/Cba₁₀-X₁₁-Ala₁₂ (SEQ ID NO: 4),where each X is an amino acid:

each D is independently an amino acid:

each E is independently an amino acid, for example an amino acidselected from Ala (alanine), D-Ala (D-alanine), Aib (α-aminoisobutyricacid), Sar (N-methyl glycine), and Ser (serine);

each R₁ and R₂ are independently —H, alkyl, alkenyl, alkynyl, arylalkyl,cycloalkyl, cycloalkylalkyl, heteroalkyl, or heterocycloalkyl,unsubstituted or substituted with halo-; or at least one of R₁ and R₂forms a macrocycle-forming linker L′ connected to the alpha position ofone of said D or E amino acids;

each L or L′ is independently a macrocycle-forming linker;

each R₅ is independently halogen, alkyl, —OR₆, —N(R₆)₂, —SR₆, —SOR₆,—SO₂R₆, —CO₂R₆, a fluorescent moiety, a radioisotope or a therapeuticagent;

each R₆ is independently —H, alkyl, alkenyl, alkynyl, arylalkyl,cycloalkylalkyl, heterocycloalkyl, a fluorescent moiety, a radioisotopeor a therapeutic agent:

each R₇ is independently —H, alkyl, alkenyl, alkynyl, arylalkyl,cycloalkyl, heteroalkyl, cycloalkylalkyl, heterocycloalkyl, cycloaryl,or heterocycloaryl, optionally substituted with R₅, or part of a cyclicstructure with a D residue:

each R₈ is independently —H, alkyl, alkenyl, alkynyl, arylalkyl,cycloalkyl, heteroalkyl, cycloalkylalkyl, heterocycloalkyl, cycloaryl,or heterocycloaryl, optionally substituted with R₅, or part of a cyclicstructure with an E residue:

v is an integer from 1-1000, for example 1-500, 1-200, 1-100, 1-50,1-30, 1-20, or 1-10;

w is an integer from 3-1000, for example 3-500, 3-200, 3-100, 3-50,3-30, 3-20, or 3-10; and.

In some embodiments of the above Formula, at least three of Xaa₃, Xaa₅,Xaa₆, Xaa₇, Xaa₈, Xaa₉, and Xaa₁₀ are the same amino acid as the aminoacid at the corresponding position of the sequencePhe₃-X₄-Glu₅-Tyr₆-Trp₇-Ala₈-Gln₉-Leu₁₀/Cba₁₀-X₁₁-Ala₁₂ (SEQ ID NO: 4).In other embodiments of the above Formula, at least four of Xaa₃, Xaa₅,Xaa₆, Xaa₇, Xaa₈, Xaa₉, and Xaa₁₀ are the same amino acid as the aminoacid at the corresponding position of the sequencePhe₃-X₄-Glu₅-Tyr₆-Trp₇-Ala₈-Gln₉-Leu₁₀/Cba₁₀-X₁₁-Ala₁₂ (SEQ ID NO: 4).Inother embodiments of the above Formula, at least five of Xaa₃, Xaa₅,Xaa₆, Xaa₇, Xaa₈, Xaa₉, and Xaa₁₀ are the same amino acid as the aminoacid at the corresponding position of the sequencePhe₃-X₄-Glu₅-Tyr₆-Trp₇-Ala₈-Gln₉-Leu₁₀/Cba₁₀-X₁₁-Ala₁₂ (SEQ ID NO: 4).In other embodiments of the above Formula, at least six of Xaa₃, Xaa₅,Xaa₆, Xaa₇, Xaa₈, Xaa₉, and Xaa₁₀ are the same amino acid as the aminoacid at the corresponding position of the sequencePhe₃-X₄-Glu₅-Tyr₆-Trp₇-Ala₈-Gln₉-Leu₁₀/Cba₁₀-X₁₁-Ala₁₂ (SEQ ID NO: 4) Inother embodiments of the above Formula, at least seven of Xaa₃, Xaa₅,Xaa₆, Xaa₇, Xaa₈, Xaa₉, and Xaa₁₀ are the same amino acid as the aminoacid at the corresponding position of the sequencePhe₃-X₄-Glu₅-Tyr₆-Trp₇-Ala₈-Gln₉-Leu₁₀/Cba₁₀-X₁₁-Ala₁₂ (SEQ ID NO: 4).

In some embodiments, w is an integer from 3-10, for example 3-6, 3-8,6-8, or 6-10. In some embodiments, w is 3. In other embodiments, w is 6.In some embodiments, v is an integer from 1-10, for example 2-5. In someembodiments, v is 2.

In one embodiment, the peptidomimetic macrocycle of Formula (I) isFormula (Ia):

or a pharmaceutically-acceptable salt thereof wherein:

each of Xaa₆, Xaa₇, Xaa₈, Xaa₁₀, Xaa₁₁, Xaa₁₂, and Xaa₁₃ isindependently an amino acid, wherein at least three, four, five, or eachof Xaa₆, Xaa₇, Xaa₈, Xaa₁₀, Xaa₁₁, Xaa₁₂, are the same amino acid as theamino acid at the corresponding position of the sequenceX₅-Thr₆-Leu₇-Leu₈-X₉-Leu₁₀-Lys₁₁/Ala₁₁-Val₁₂/Ala₁₂ (SEQ ID NO: 6), whereeach of X₅ and X₉ is independently an amino acid.

In some embodiments, the peptidomimetic macrocycle of Formula (Ia) isFormula (Ia-1):

or a pharmaceutically-acceptable salt thereof, wherein each Xaa₁₄ isindependently an amino acid.

In some embodiments, the peptidomimetic macrocycle of Formula (Ia) isFormula (Ia-2):

or a pharmaceutically-acceptable salt thereof, wherein each Xaa₁₄ andXaa₁₅ is independently an amino acid.

In one embodiment, the peptidomimetic macrocycle of Formula (I) isFormula (Ib):

or a pharmaceutically-acceptable salt thereof, wherein: each of Xaa₆,Xaa₇, Xaa₈, Xaa₉, Xaa₁₀, Xaa₁₁ and Xaa₁₃ is independently an amino acid,wherein at least three, four, five, or each of Xaa₆, Xaa₇, Xaa₈, Xaa₉,Xaa₁₀, and Xaa₁₁ are the same amino acid as the amino acid at thecorresponding position of the sequenceX₅-Thr₆-Leu₇-Leu₈-Phe₉-Leu₁₀-Lys₁₁/Ala₁₁-X₁₂ (SEQ ID NO: 7), where eachof X₅ and X₁₂ is independently an amino acid.

In some embodiments, the peptidomimetic macrocycle of Formula (Ib) isFormula (Ib-1):

or a pharmaceutically-acceptable salt thereof, wherein each Xaa₁₄ isindependently an amino acid.

In some embodiments, the peptidomimetic macrocycle of Formula (Ib) isFormula (Ib-2):

or a pharmaceutically-acceptable salt thereof, wherein each Xaa₁₄ andXaa₁₅ is independently an amino acid.

In some embodiments, the invention provides a peptidomimetic macrocycleof Formula (IX):

wherein the peptidomimetic macrocycle binds MCL-1 selectively overanother protein that has a BH3 domain. wherein:

each A, C, D, and E is independently a natural or non-natural aminoacid;

each B is independently a natural or non-natural amino acid, amino acidanalog

[—NH-L₃-CO—], [—NH-L₃-SO₂—], or [—NH-L₃-];

each L is independently a macrocycle-forming linker;

each L′ is independently alkylene, alkenylene, alkynylene,heteroalkylene, cycloalkylene, heterocycloalkylene, arylene, orheteroarylene, each being optionally substituted with R₅, or a bond, ortogether with R₁ and the atom to which both R₁ and L′ are bound forms aring;

each L″ is independently alkylene, alkenylene, alkynylene,heteroalkylene, cycloalkylene, heterocycloalkylene, arylene, orheteroarylene, each being optionally substituted with R₅, or a bond, ortogether with R₂ and the atom to which both R₂ and L″ are bound forms aring;

each R₁ is independently —H, alkyl, alkenyl, alkynyl, arylalkyl,cycloalkyl, cycloalkylalkyl, heteroalkyl, or heterocycloalkyl,unsubstituted or substituted with halo-, or together with L′ and theatom to which both R₁ and L′ are bound forms a ring;

each R₂ is independently —H, alkyl, alkenyl, alkynyl, arylalkyl,cycloalkyl, cycloalkylalkyl, heteroalkyl, or heterocycloalkyl,unsubstituted or substituted with halo-, or together with L″ and theatom to which both R₂ and L″ are bound forms a ring;

each R₃ is independently hydrogen. alkyl, alkenyl, alkynyl, arylalkyl,heteroalkyl, cycloalkyl, heterocycloalkyl, cycloalkylalkyl, aryl, orheteroaryl, optionally substituted with R₅;

each L₃ is independently alkylene, alkenylene, alkynylene,heteroalkylene, cycloalkylene, heterocycloalkylene, arylene,heteroarylene, or [—R₄—K—R₄—]. each being optionally substituted withR₅;

each R₄ is independently alkylene, alkenylene, alkynylene,heteroalkylene, cycloalkylene, heterocycloalkylene, arylene, orheteroarylene;

each K is independently O, S, SO, SO₂, CO, CO₂, or CONR₃;

each n is independently an integer from 1-5;

each R₅ is independently halogen, alkyl, —OR₆, —N(R)₂, —SR₆, —SOR₆,—SO₂R₆, —CO₂R₆, a fluorescent moiety, a radioisotope, or a therapeuticagent;

each R₆ is independently —H, alkyl, alkenyl, alkynyl, arylalkyl,cycloalkylalkyl, heterocycloalkyl, a fluorescent moiety. a radioisotope,or a therapeutic agent;

each R₇ is independently —H, alkyl, alkenyl, alkynyl, arylalkyl,cycloalkyl, heteroalkyl, cycloalkylalkyl, heterocycloalkyl, aryl, orheteroaryl, optionally substituted with R₅, or part of a cyclicstructure with a D residue;

each R₈ is independently —H, alkyl, alkenyl, alkynyl, arylalkyl,cycloalkyl, heteroalkyl, cycloalkylalkyl, heterocycloalkyl, aryl, orheteroaryl, optionally substituted with R₅, or part of a cyclicstructure with an E residue;

each v and w is independently an integer from 1-1000;

u is an integer from 1-10; and

each x, y and z is independently an integer from 0-10, or apharmaceutically-acceptable salt thereof.

In some embodiments, the invention provides a peptidomimetic macrocyclehaving the formula (SEQ ID NO: 8):

wherein:

each D and E is independently an amino acid residue;

each R¹ and R² are independently alkyl, alkenyl, alkynyl, arylalkyl,cycloalkyl, cycloalkylalkyl, heteroalkyl, or heterocycloalkyl, eachbeing optionally substituted with halo-; —H, or at least one of R¹ andR² forms a macrocycle-forming linker L′ connected to the alpha positionof one of the D or E amino acid residues;

each L is a macrocycle-forming linker of the formula -L¹-L²- or-L¹-L²-L³-;

each L¹, L², and L³ are independently alkylene, alkenylene, alkynylene,heteroalkylene, cycloalkylene, heterocycloalkylene, arylene,heteroarylene, or [—R⁴—K—R⁴—]_(n), each being optionally substitutedwith R⁵;

each R³ is independently —H, alkyl, alkenyl, alkynyl, arylalkyl,cycloalkyl, heteroalkyl, cycloalkylalkyl, heterocycloalkyl, aryl, orheteroaryl, each being optionally substituted with R;

each R⁴ is independently alkylene, alkenylene, alkynylene,heteroalkylene, cycloalkylene, heterocycloalkylene, arylene, orheteroarylene, each being optionally substituted with R⁵;

each K is independently O, S, SO. SO₂, CO, CO₂, or CONR³;

each R⁵ is independently halogen, alkyl, —OR⁶, —N(R⁶)₂, —SR⁶, —SOR⁶,—SO₂R⁶, —CO₂R⁶, a fluorescent moiety, a radioisotope, or a therapeuticagent;

each R⁶ is independently —H, alkyl, alkenyl, alkynyl, arylalkyl,cycloalkylalkyl, heterocycloalkyl, a fluorescent moiety, a radioisotope,or a therapeutic agent;

R⁷ is —H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, heteroalkyl,cycloalkylalkyl, heterocycloalkyl, aryl, or heteroaryl, each beingoptionally substituted with R, or part of a cyclic structure with a Dresidue;

R⁸ is —H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, heteroalkyl,cycloalkylalkyl, heterocycloalkyl, aryl, or heteroaryl, each beingoptionally substituted with R, or part of a cyclic structure with an Eresidue;

each of Xaa¹ and Xaa² is independently an amino acid residue or absent:

Xaa³ is Ala, Aib, Asp, Asn, Cys, Glu, Gln, His, Ile, Lys, Leu, Met, Arg,Ser, Thr, Val, Trp, Tyr, or an analog of any of the foregoing;

v is an integer from 1-1000:

w is an integer from 0-1000; and

n is an integer from 1-5, or

a pharmaceutically-acceptable salt thereof.

In some embodiments, the invention provides a peptidomimetic macrocycleof the formula:

wherein:

each D and E is independently an amino acid residue;

R¹ and R² are independently alkyl, alkenyl, alkynyl, arylalkyl,cycloalkyl, cycloalkylalkyl, heteroalkyl, or heterocycloalkyl, eachbeing optionally substituted with halo-; —H, or at least one of R¹ andR² forms a macrocycle-forming linker L′ connected to the alpha positionof one of the D or E amino acid residues;

each L or L′ is independently a macrocycle-forming linker of the formula-L¹-L²- or -L¹-L-L-; L¹, L², and L are independently alkylene,alkenylene, alkynylene, heteroalkylene, cycloalkylene.heterocycloalkylene, arylene, heteroarylene, or [—R⁴—K—R⁴—]_(n), eachbeing optionally substituted with R⁵;

each R³ is independently —H, alkyl, alkenyl, alkynyl, arylalkyl,cycloalkyl, heteroalkyl, cycloalkylalkyl, heterocycloalkyl, aryl, orheteroaryl, each being optionally substituted with R;

each R⁴ is independently alkylene, alkenylene, alkynylene,heteroalkylene, cycloalkylene, heterocycloalkylene, arylene, orheteroarylene, each being optionally substituted with R;

each K is independently O, S, SO, SO₂, CO, CO₂, or CONR³;

each R⁵ is independently halogen, alkyl, —OR⁶, —N(R⁶)₂, —SR⁶, —SOR⁶,—SO₂R⁶, —CO₂R⁶, a fluorescent moiety, a radioisotope, or a therapeuticagent;

each R⁶ is independently —H, alkyl, alkenyl, alkynyl, arylalkyl,cycloalkylalkyl, heterocycloalkyl, a fluorescent moiety. a radioisotope,or a therapeutic agent;

R⁷ is —H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, heteroalkyl,cycloalkylalkyl, heterocycloalkyl, aryl, or heteroaryl, each beingoptionally substituted with R, or part of a cyclic structure with a Dresidue;

R⁸ is —H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, heteroalkyl,cycloalkylalkyl, heterocycloalkyl. aryl, or heteroaryl, each beingoptionally substituted with R⁵, or part of a cyclic structure with an Eresidue;

each of Xaa¹ and Xaa² is independently an amino acid residue or absent:

v is an integer from 1-1000;

w is an integer from 0-1000; and

n is an integer from 1-5, or

a pharmaceutically-acceptable salt thereof.

In some embodiments, the invention provides a peptidomimetic macrocyclecomprising an amino acid sequence of formula:

X₁-X₂-X₃-X₄-X₅-X₆-X₇-X₈-X₉-X₁₀-X₁₁-X₁₂-X₁₃-X₁₄-X₁₅-X₁₆-X₁₇-X₁₈-X₁₉-X₂₀-X₂₁

wherein:

X₁ is Ile, Arg, Ala, Lys, Pro, Leu, Asp, Glu, His, Ser, Gln, Phe, ananalog thereof, or absent;

X₂ is Trp, Arg, Ala, Asn, Phe, Pro, Leu, Ser, Lys, Tyr, His, Cou, Cou2,Cou4, Cou7, an analog thereof, a crosslinked amino acid, or absent;

X3 is Ile, Ala, Leu, Phe, Tyr, Val, Asp, Trp, Pro, Gln, Chg, Ac5c, Ac6c,Tha, Bip, Cha, Adm, hCha, an analog thereof, or absent;

X₄ is Ala, Gln, Asp, Val, Gly. Ser, Leu, Phe, Cha, A4, an analog,thereof, a crosslinked amino acid, or absent;

X₅ is Gln, Ala, Leu, Phe, Tyr, Gly, Ile, Val, Arg, Glu, Pro, Asp, MO,MO2, an analog thereof, a crosslinked amino acid, or absent;

X₆ is Glu, Gln, His, Ala, Ser, Arg, Ile, Leu, Thr, Phe, Val, Tyr, Gly,Nle, St, an analog thereof, or absent;

X₇ is Ala, Leu, Phe, Ile, 2Nal, 1Nal, 3cf, Chg. Cha, Adm, hCha, Igl,Bip, an analog thereof, or absent;

X₈ is Arg, Ala, Asp, Glu, Thr, His, Gln, Gly, Asn, Phe, Cit, St, ananalog thereof, a crosslinked amino acid, or absent;

X₉ is Arg, Ala, Asp, Lys, Asn, Gly, Ser, Gln, Cys, Nle, St, an analogthereof, or a crosslinked amino acid;

X₁₀ is Ile, Val, Ala, Asp, Asn, Phe, Tba, hL, hhL, Nle, Chg, Cha, ananalog thereof, or a crosslinked amino acid;

X₁₁ is Gly, Val, Ala. Leu, Ile, Asp, Glu, Cha, Aib, Abu, an analogthereof, or a crosslinked amino acid;

X₁₂ is Asp, Ala, Asn, Gly, Arg, Glu, Lys, Leu, Ne, an analog thereof, ora crosslinked amino acid;

X₁₃ is Ala, Glu, Gln, Leu, Lys, Asp, Tyr, lie, Ser, Cys, St, Sta5, Aib,Ne, an analog thereof, or a crosslinked amino acid;

X₁₄ is Phe, Ala, Leu, Val, Tyr, Glu, His, Ile, Nle, 1Nal, 2Nal, Chg,Cha, BiP, an analog thereof, or a crosslinked amino acid;

X₁₅ is Asn, Gln, Ser, His, Glu, Asp, Ala, Leu, lie, St, Ne, Aib, ananalog thereof, a crosslinked amino acid, or absent;

X₁₆ is Ala, Glu, Asp, Arg, Lys, Phe, Gly, Gln, Aib, Cha, St, an analogthereof, a crosslinked amino acid, or absent;

X₁₇ is Phe, Tyr, Ala, Leu, Asn, Ser, Gln, Arg, His, Thr, Cou2, Cou3,Cou7, Dpr, Amf, Damf, Amye, an analog thereof, a crosslinked amino acid,or absent;

X₁₈ is Tyr, Ala. Ile, Phe, His, Arg, Lys, Trp, Orn, Amf, Amyc, Cha,2Nal, an analog thereof, or absent;

X₁₉ is Ala, Lys, Arg, His, Ser, Gln, Glu, Asp, Thr, Aib, Cha, an analogthereof, a crosslinked amino acid, or absent; and

X₂₀ is Arg, His, Ala, Thr, Lys, Amr, an analog thereof, a crosslinkedamino acid, or absent; and

X₂₁ is Arg, His, Ala, Amr, an analog thereof, or absent, or

a pharmaceutically-acceptable salt thereof,

wherein at least two of the amino acids of the amino acid sequence are acrosslinked amino acid.

In some embodiments, the invention provides a peptidomimetic macrocyclecomprising an amino acid sequence with C-terminal amino acid residuesthat are -His-His, wherein the peptidomimetic macrocycle comprises acrosslink connecting at least two amino acid residues, or apharmaceutically-acceptable salt thereof. In an embodiment of any of theFormulas described herein, of the macrocycle-forming linker (L or L′)has a formula -L₁-L₂-, wherein

L₁ and L₂ are independently alkylene, alkenylene, alkynylene,heteroalkylene, cycloalkylene, heterocycloalkylene, cycloarylene,heterocycloarylene, or [—R₄—K—R₄-], each being optionally substitutedwith R₅;each R₄ is independently alkylene, alkenylene, alkynylene,heteroalkylene, cycloalkylene, heterocycloalkylene, arylene, orheteroarylene;each K is independently O, S, SO, SO₂, CO, CO₂, or CONR₃;each R₃ independently is hydrogen, alkyl, alkenyl, alkynyl, arylalkyl,heteroalkyl, cycloalkyl, heterocycloalkyl, cycloalkylalkyl, cycloaryl,or heterocycloaryl, optionally substituted with R₅; andn is an integer from 1-5.

In some embodiments, L (or L′) is a macrocycle-forming linker of theformula

Exemplary embodiments of such macrocycle-forming linkers L are shownbelow.

In an embodiment of any of the Formulas described herein, L₁ and L₂,either alone or in combination, form a triazole or a thioether.

In an embodiment of any of the Formulas described herein, L₁ and L₂,either alone or in combination, do not form a triazole or a thioether.

In one example, at least one of R₁ and R₂ is alkyl, unsubstituted orsubstituted with halo-. In another example, both R₁ and R₂ areindependently alkyl, unsubstituted or substituted with halo-. In someembodiments, at least one of R₁ and R₂ is methyl. In other embodiments,R₁ and R₂ are methyl.

In some embodiments, x+y+z is at least 3. In other embodiments, x+y+z is1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In some embodiments, the sum of x+y+zis 3 or 6. In some embodiments, the sum of x+y+z is 3. In otherembodiments, the sum of x+v+z is 6. Each occurrence of A, B, C. D or Ein a macrocycle or macrocycle precursor is independently selected. Forexample, a sequence represented by the formula [A]_(x), when x is 3,encompasses embodiments where the amino acids are not identical, e.g.Gln-Asp-Ala as well as embodiments where the amino acids are identical,e.g. Gln-Gln-Gln. This applies for any value of x, y, or z in theindicated ranges. Similarly, when u is greater than 1, each compound canencompass peptidomimetic macrocycles which are the same or different.For example, a compound can comprise peptidomimetic macrocyclescomprising different linker lengths or chemical compositions.

In some embodiments, the peptidomimetic macrocycle comprises a secondarystructure which is an α-helix and R_(a) is —H, allowing intrahelicalhydrogen bonding. In some embodiments, at least one of A, B, C, D or Eis an α,α-disubstituted amino acid. In one example, B is anα,α-disubstituted amino acid. For instance, at least one of A, B, C, Dor E is 2-aminoisobutyric acid. In other embodiments, at least one of A,B, C, D or E is

In other embodiments, the length of the macrocycle-forming linker L asmeasured from a first Cα to a second Cα is selected to stabilize adesired secondary peptide structure, such as an α-helix formed byresidues of the peptidomimetic macrocycle including, but not necessarilylimited to, those between the first Cα to a second Cα.

In some embodiments. a peptidomimetic macrocycle of Formula (I) hasFormula:

wherein:each A, C, D, and E is independently a natural or non-natural aminoacid:each B is independently a natural or non-natural amino acid, amino acidanalog,

[—NH-L₃-CO—], [—NH-L-SO₂—], or [—NH-L₃-]each L is independently a macrocycle-forming linker;each L′ is independently alkylene, alkenylene, alkynylene,heteroalkylene, cycloalkylene, heterocycloalkylene, arylene, orheteroarylene, each being optionally substituted with R₅, or a bond, ortogether with R₁ and the atom to which both R₁ and L′ are bound forms aring;each L″ is independently alkylene, alkenylene, alkynylene,heteroalkylene, cycloalkylene, heterocycloalkylene, arylene, orheteroarylene, each being optionally substituted with R₅, or a bond, ortogether with R₂ and the atom to which both R₂ and L′ are bound forms aring;each R₁ is independently —H, alkyl, alkenyl, alkynyl, arylalkyl,cycloalkyl, cycloalkylalkyl, heteroalkyl, or heterocycloalkyl,unsubstituted or substituted with halo-, or together with L′ and theatom to which both R₁ and L′ are bound forms a ring;each R₂ is independently —H, alkyl, alkenyl, alkynyl, arylalkyl,cycloalkyl, cycloalkylalkyl, heteroalkyl, or heterocycloalkyl,unsubstituted or substituted with halo-, or together with L″ and theatom to which both R₂ and L″ are bound forms a ring;each R₃ is independently hydrogen, alkyl, alkenyl, alkynyl, arylalkyl,heteroalkyl, cycloalkyl, heterocycloalkyl, cycloalkylalkyl, aryl, orheteroaryl, optionally substituted with R₅;each L₃ is independently alkylene, alkenylene, alkynylene,heteroalkylene, cycloalkylene, heterocycloalkylene, arylene,heteroarylene, or [—R₄—K—R₄—]_(n), each being optionally substitutedwith R₅;each R₄ is alkylene, alkenylene, alkynylene, heteroalkylene,cycloalkylene, heterocycloalkylene, arylene, or heteroarylene;each K is independently O, S, SO, SO₂, CO, CO₂, or CONR₃;each n is independently an integer from 1-5;each R₅ is independently halogen, alkyl, —OR₆, —N(R₆)₂, —SR₆, —SOR₆,—SO₂R₆, —CO₂R₆, a fluorescent moiety, a radioisotope or a therapeuticagent;each R₆ is independently —H, alkyl, alkenyl, alkynyl, arylalkyl,cycloalkylalkyl, heterocycloalkyl, a fluorescent moiety, a radioisotopeor a therapeutic agent;each R₇ is independently —H, alkyl, alkenyl, alkynyl, arylalkyl,cycloalkyl, heteroalkyl, cycloalkylalkyl, heterocycloalkyl, aryl, orheteroaryl, optionally substituted with R₅, or part of a cyclicstructure with a D residue;

each R₈ is independently —H, alkyl, alkenyl, alkynyl, arylalkyl,cycloalkyl, heteroalkyl, cycloalkylalkyl, heterocycloalkyl, aryl, orheteroaryl, optionally substituted with R₅, or part of a cyclicstructure with an E residue;

each v and w is independently an integer from 1-1000, for example 1-500,1-200, 1-100, 1-50, 1-40, 1-25, 1-20, 1-15, or 1-10; andeach u, x, y and z is independently an integer from 0-10.

In some embodiments, the peptidomimetic macrocycles have the Formula I:

wherein:each A, C, D, and E is independently a natural or non-natural aminoacid;each B is independently a natural or non-natural amino acid, amino acidanalog,

[—NH-L₃-CO—], [—NH-L₃-SO₂—], or [—NH-L₃-];each R₁ and R₂ is independently —H, alkyl, alkenyl, alkynyl. arylalkyl,cycloalkyl, cycloalkylalkyl, heteroalkyl, or heterocycloalkyl,unsubstituted or substituted with halo-;each R₃ is independently hydrogen, alkyl, alkenyl, alkynyl, arylalkyl,heteroalkyl, cycloalkyl, heterocycloalkyl, cycloalkylalkyl, aryl, orheteroaryl, optionally substituted with R₅;each L is independently a macrocycle-forming linker of the formula

each L₁, L₂ and L₃ is independently alkylene, alkenylene, alkynylene,heteroalkylene, cycloalkylene, heterocycloalkylene, arylene,heteroarylene, or [—R₄—K—R₄—]_(n), each being optionally substitutedwith R₅;each R₄ is alkylene, alkenylene, alkynylene, heteroalkylene,cycloalkylene, heterocycloalkylene, arylene, or heteroarylene;each K is independently O, S, SO, SO₂, CO, CO₂, or CONR₃;each R₅ is independently halogen, alkyl, —OR₆, —N(R₆)₂, —SR₆, —SOR₆,—SO₂R₆, —CO₂R₆, a fluorescent moiety, a radioisotope or a therapeuticagent;each R₆ is independently —H, alkyl, alkenyl, alkynyl, arylalkyl,cycloalkylalkyl, heterocycloalkyl, a fluorescent moiety, a radioisotopeor a therapeutic agent:each R₇ is independently —H, alkyl, alkenyl, alkynyl, arylalkyl,cycloalkyl, heteroalkyl, cycloalkylalkyl, heterocycloalkyl, aryl, orheteroaryl, optionally substituted with R₅, or part of a cyclicstructure with a D residue;each R₈ is independently —H, alkyl, alkenyl, alkynyl, arylalkyl,cycloalkyl, heteroalkyl, cycloalkylalkyl, heterocycloalkyl, aryl, orheteroaryl, optionally substituted with R₅, or part of a cyclicstructure with an E residue;each v and w is independently an integer from 1-1000;each u, x, y and z is independently integers from 0-10; andn is an integer from 1-5.

In one embodiment, the peptidomimetic macrocycle of Formula (I) is:

wherein each R₁ and R₂ is independently —H. alkyl, alkenyl, alkynyl,arylalkyl, cycloalkyl, cycloalkylalkyl, heteroalkyl, orheterocycloalkyl, unsubstituted or substituted with halo-.

In related embodiments, the peptidomimetic macrocycle of Formula (I) is:

wherein each R₁′ and R₂′ is independently an amino acid.In other embodiments, the peptidomimetic macrocycle of Formula (I) is acompound of any of the formulas shown below:

wherein “AA” represents any natural or non-natural amino acid side chainand “

” is [D]_(v), [E]_(w) as defined above, and n is an integer between 0and 20, 50, 100, 200, 300, 400 or 500. In some embodiments, n is 0. Inother embodiments, n is less than 50.

Exemplary embodiments of the macrocycle-forming linker L are shownbelow.

In other embodiments, D and/or E in the compound of Formula I arefurther modified in order to facilitate cellular uptake. In someembodiments, lipidating or PEGylating a peptidomimetic macrocyclefacilitates cellular uptake, increases bioavailability, increases bloodcirculation, alters pharmacokinetics, decreases immunogenicity and/ordecreases the needed frequency of administration.

In other embodiments, at least one of [D] and [E] in the compound ofFormula I represents a moiety comprising an additionalmacrocycle-forming linker such that the peptidomimetic macrocyclecomprises at least two macrocycle-forming linkers. In a specificembodiment, a peptidomimetic macrocycle comprises two macrocycle-forminglinkers. In an embodiment, u is 2.

In some embodiments, any of the macrocycle-forming linkers describedherein can be used in any combination with any of the sequences shown inTable 1, Table 1a, Table 1b, and Table 1c and also with any of theR-substituents indicated herein.

In some embodiments, the peptidomimetic macrocycle comprises at leastone α-helix motif. For example, A, B and/or C in the compound of FormulaI include one or more α-helices. As a general matter, α-helices includebetween 3 and 4 amino acid residues per turn. In some embodiments, theα-helix of the peptidomimetic macrocycle includes 1 to 5 turns and,therefore, 3 to 20 amino acid residues. In specific embodiments, theα-helix includes 1 turn, 2 turns, 3 turns, 4 turns, or 5 turns. In someembodiments, the macrocycle-forming linker stabilizes an α-helix motifincluded within the peptidomimetic macrocycle. Thus, in someembodiments, the length of the macrocycle-forming linker L from a firstCa to a second Ca is selected to increase the stability of an α-helix.In some embodiments, the macrocycle-forming linker spans from 1 turn to5 turns of the α-helix. In some embodiments, the macrocycle-forminglinker spans approximately 1 turn, 2 turns, 3 turns, 4 turns, or 5 turnsof the α-helix. In some embodiments, the length of themacrocycle-forming linker is approximately 5 Å to 9 Å per turn of theα-helix, or approximately 6 Å to 8 Å per turn of the α-helix. Where themacrocycle-forming linker spans approximately 1 turn of an α-helix, thelength is equal to approximately 5 carbon-carbon bonds to 13carbon-carbon bonds, approximately 7 carbon-carbon bonds to 11carbon-carbon bonds, or approximately 9 carbon-carbon bonds. Where themacrocycle-forming linker spans approximately 2 turns of an α-helix, thelength is equal to approximately 8 carbon-carbon bonds to 16carbon-carbon bonds, approximately 10 carbon-carbon bonds to 14carbon-carbon bonds, or approximately 12 carbon-carbon bonds. Where themacrocycle-forming linker spans approximately 3 turns of an α-helix, thelength is equal to approximately 14 carbon-carbon bonds to 22carbon-carbon bonds, approximately 16 carbon-carbon bonds to 20carbon-carbon bonds, or approximately 18 carbon-carbon bonds. Where themacrocycle-forming linker spans approximately 4 turns of an α-helix, thelength is equal to approximately 20 carbon-carbon bonds to 28carbon-carbon bonds, approximately 22 carbon-carbon bonds to 26carbon-carbon bonds, or approximately 24 carbon-carbon bonds. Where themacrocycle-forming linker spans approximately 5 turns of an α-helix, thelength is equal to approximately 26 carbon-carbon bonds to 34carbon-carbon bonds, approximately 28 carbon-carbon bonds to 32carbon-carbon bonds, or approximately 30 carbon-carbon bonds. Where themacrocycle-forming linker spans approximately 1 turn of an α-helix, thelinkage contains approximately 4 atoms to 12 atoms, approximately 6atoms to 10 atoms, or approximately 8 atoms. Where themacrocycle-forming linker spans approximately 2 turns of the α-helix,the linkage contains approximately 7 atoms to 15 atoms, approximately 9atoms to 13 atoms, or approximately 11 atoms. Where themacrocycle-forming linker spans approximately 3 turns of the α-helix,the linkage contains approximately 13 atoms to 21 atoms, approximately15 atoms to 19 atoms, or approximately 17 atoms. Where themacrocycle-forming linker spans approximately 4 turns of the α-helix,the linkage contains approximately 19 atoms to 27 atoms, approximately21 atoms to 25 atoms, or approximately 23 atoms. Where themacrocycle-forming linker spans approximately 5 turns of the α-helix,the linkage contains approximately 25 atoms to 33 atoms, approximately27 atoms to 31 atoms, or approximately 29 atoms. Where themacrocycle-forming linker spans approximately 1 turn of the α-helix, theresulting macrocycle forms a ring containing approximately 17 members to25 members, approximately 19 members to 23 members, or approximately 21members. Where the macrocycle-forming linker spans approximately 2 turnsof the α-helix, the resulting macrocycle forms a ring containingapproximately 29 members to 37 members, approximately 31 members to 35members, or approximately 33 members. Where the macrocycle-forminglinker spans approximately 3 turns of the α-helix, the resultingmacrocycle forms a ring containing approximately 44 members to 52members, approximately 46 members to 50 members, or approximately 48members. Where the macrocycle-forming linker spans approximately 4 turnsof the α-helix, the resulting macrocycle forms a ring containingapproximately 59 members to 67 members, approximately 61 members to 65members, or approximately 63 members. Where the macrocycle-forminglinker spans approximately 5 turns of the α-helix, the resultingmacrocycle forms a ring containing approximately 74 members to 82members, approximately 76 members to 80 members, or approximately 78members.

In other embodiments, provided are peptidomimetic macrocycles of Formula(IV) or (IVa):

wherein:each A, C, D, and E is independently a natural or non-natural aminoacid, and the terminal D and E independently optionally include acapping group;each B is independently a natural or non-natural amino acid, amino acidanalog,

[—NH-L₃-CO—], [—NH-L₃-SO₂—], or [—NH-L₃-];each R₁ and R₂ are independently —H, alkyl, alkenyl, alkynyl, arylalkyl,cycloalkyl, cycloalkylalkyl, heteroalkyl, or heterocycloalkyl,unsubstituted or substituted with halo-; or at least one of R₁ and R₂forms a macrocycle-forming linker L′ connected to the alpha position ofone of said D or E amino acids;each R₃ independently is hydrogen, alkyl, alkenyl, alkynyl, arylalkyl,heteroalkyl, cycloalkyl, heterocycloalkyl, cycloalkylalkyl, cycloaryl,or heterocycloaryl, optionally substituted with R₅;each L independently is a macrocycle-forming linker of the formula-L₁-L₂-;each L₁, L₂ and L₃ are independently alkylene, alkenylene, alkynylene,heteroalkylene, cycloalkylene, heterocycloalkylene, cycloarylene,heterocycloarylene, or [—R₄—K—R₄—]_(n), each being optionallysubstituted with R₅;

each R₄ is independently alkylene, alkenylene, alkynylene,heteroalkylene, cycloalkylene, heterocycloalkylene, arylene, orheteroarylene;

each K is independently O, S, SO, SO₂ CO, CO₂, or CONR₃;each R₅ is independently halogen, alkyl, —OR₆, —N(R₆)₂, —SR₆, —SOR₆,—SO₂R₆, —CO₂R₆, a fluorescent moiety, a radioisotope or a therapeuticagent;each R₆ is independently —H, alkyl, alkenyl, alkynyl, arylalkyl,cycloalkylalkyl, heterocycloalkyl, a fluorescent moiety, a radioisotopeor a therapeutic agent;each R₇ is independently —H, alkyl, alkenyl, alkynyl, arylalkyl,cycloalkyl, heteroalkyl, cycloalkylalkyl, heterocycloalkyl, cycloaryl,or heterocycloaryl, optionally substituted with R₅;each v and w are independently integers from 1-1000;u is an integer from 1-10;each x, y and z are independently integers from 0-10; andeach n independently is an integer from 1-5.

In one example, L₁ and L₂, either alone or in combination, do not form atriazole or a thioether.

In one example, at least one of R₁ and R₂ is alkyl, unsubstituted orsubstituted with halo-. In another example, both R₁ and R₂ areindependently alkyl, unsubstituted or substituted with halo-. In someembodiments, at least one of R₁ and R₂ is methyl. In other embodiments,R₁ and R₂ are methyl.

In some embodiments, x+v+z is at least 1. In other embodiments, x+y+z isat least 2. In other embodiments, x+y+z is 1, 2, 3, 4, 5, 6, 7, 8, 9 or10. Each occurrence of A, B, C, D or E in a macrocycle or macrocycleprecursor is independently selected. For example, a sequence representedby the formula [A]_(x), when x is 3, encompasses embodiments where theamino acids are not identical, e.g. Gin-Asp-Ala as well as embodimentswhere the amino acids are identical. e.g. Gln-Gln-Gln. This applies forany value of x, y, or z in the indicated ranges.

In some embodiments, the peptidomimetic macrocycle comprises a secondarystructure which is an α-helix and R₈ is —H, allowing intrahelicalhydrogen bonding. In some embodiments, at least one of A, B, C, D or Eis an α,α-disubstituted amino acid. In one example, B is anα,α-disubstituted amino acid. For instance, at least one of A, B, C, Dor E is 2-aminoisobutyric acid. In other embodiments, at least one of A,B, C, D or E is

In other embodiments, the length of the macrocycle-forming linker L asmeasured from a first Cα to a second Cα is selected to stabilize adesired secondary peptide structure, such as an α-helix formed byresidues of the peptidomimetic macrocycle including, but not necessarilylimited to, those between the first Cα to a second Cα.

Exemplary embodiments of the macrocycle-forming linker -L₁-L₂- are shownbelow.

In some embodiments, L is a macrocycle-forming linker of the formula

Exemplary embodiments of such macrocycle-forming linkers L are shownbelow.

Unless otherwise stated, any compounds (including peptidomimeticmacrocycles, macrocycle precursors, and other compositions) are alsomeant to encompass compounds which differ only in the presence of one ormore isotopically enriched atoms. For example, compounds having thedescribed structures except for the replacement of a hydrogen by adeuterium or tritium, or the replacement of a carbon by ¹³C- or¹⁴C-enriched carbon are within the scope of this disclosure.

In some embodiments, the compounds disclosed herein can containunnatural proportions of atomic isotopes at one or more of atoms thatconstitute such compounds. For example, the compounds can beradiolabeled with radioactive isotopes, such as for example tritium(3H), iodine-125 (¹²⁵I) or carbon-14 (¹⁴C). In other embodiments, one ormore carbon atoms is replaced with a silicon atom. The compounds(including peptidomimetic macrocycles, macrocycle precursors, and othercompositions) also include salts thereof. For example, salts of acidicand basic amino acids. All isotopic variations of the compoundsdisclosed herein, whether radioactive or not, are contemplated herein.

The compound or peptidomimetic macrocycles described herein can be atleast 1% pure, at least 2% pure, at least 3% pure, at least 4% pure, atleast 5% pure, at least 6% pure, at least 7% pure, at least 8% pure, atleast 9% pure, at least 10% pure, at least 11% pure, at least 12% pure,at least 13% pure, at least 14% pure, at least 15% pure, at least 16%pure, at least 17% pure, at least 18% pure, at least 19% pure, at least20% pure, at least 21% pure, at least 22% pure, at least 23% pure, atleast 24% pure, at least 25% pure, at least 26% pure, at least 27% pure,at least 28% pure, at least 29% pure, at least 30% pure, at least 31%pure, at least 32% pure, at least 33% pure, at least 34% pure, at least35% pure, at least 36% pure, at least 37% pure, at least 38% pure, atleast 39% pure, at least 40% pure, at least 41% pure, at least 42% pure,at least 43% pure, at least 44% pure, at least 45% pure, at least 46%pure, at least 47% pure, at least 48% pure, at least 49% pure, at least50% pure, at least 51% pure, at least 52% pure, at least 53% pure, atleast 54% pure, at least 55% pure, at least 56% pure, at least 57% pure,at least 58% pure, at least 59% pure, at least 60% pure, at least 61%pure, at least 62% pure, at least 63% pure, at least 64% pure, at least65% pure, at least 66% pure, at least 67% pure, at least 68% pure, atleast 69% pure, at least 70% pure, at least 71% pure, at least 72% pure,at least 73% pure, at least 74% pure, at least 75% pure, at least 76%pure, at least 77% pure, at least 78% pure, at least 79% pure, at least80% pure, at least 81% pure, at least 82% pure, at least 83% pure, atleast 84% pure, at least 85% pure, at least 86% pure, at least 87% pure,at least 88% pure, at least 89% pure, at least 90% pure, at least 91%pure, at least 92% pure, at least 93% pure, at least 94% pure, at least95% pure, at least 96% pure, at least 97% pure, at least 98% pure, atleast 99% pure, at least 99.1% pure, at least 99.2% pure, at least 99.3%pure, at least 99.4% pure, at least 99.5% pure, at least 99.6% pure, atleast 99.7% pure, at least 99.8% pure, or at least 99.9% pure on achemical, optical. isomeric, enantiomeric, or diastereomeric basis.Purity can be assessed, for example, by HPLC, MS, LC/MS, melting point,or NMR.

Two or more peptides can share a degree of homology. A pair of peptidescan have, for example, up to about 20% pairwise homology, up to about25% pairwise homology, up to about 30% pairwise homology, up to about35% pairwise homology, up to about 40% pairwise homology, up to about45% pairwise homology, up to about 50% pairwise homology, up to about55% pairwise homology, up to about 60% pairwise homology, up to about65% pairwise homology, up to about 70% pairwise homology, up to about75% pairwise homology, up to about 80% pairwise homology, up to about85% pairwise homology, up to about 90% pairwise homology, up to about95% pairwise homology, up to about 96% pairwise homology, up to about97% pairwise homology, up to about 98% pairwise homology, up to about99% pairwise homology, up to about 99.5% pairwise homology, or up toabout 99.9% pairwise homology. A pair of peptides can have. for example,at least about 20% pairwise homology, at least about 25% pairwisehomology, at least about 30% pairwise homology, at least about 35%pairwise homology, at least about 40% pairwise homology, at least about45% pairwise homology. at least about 50% pairwise homology, at leastabout 55% pairwise homology, at least about 60% pairwise homology, atleast about 65% pairwise homology, at least about 70% pairwise homology.at least about 75% pairwise homology, at least about 80% pairwisehomology, at least about 85% pairwise homology, at least about 90%pairwise homology, at least about 95% pairwise homology, at least about96% pairwise homology, at least about 97% pairwise homology, at leastabout 98% pairwise homology, at least about 99% pairwise homology, atleast about 99.5% pairwise homology, at least about 99.9% pairwisehomology.

Various methods and software programs can be used to determine thehomology between two or more peptides, such as NCBI BLAST, Clustal W,MAFFT, Clustal Omega, AlignMe, Praline, or another suitable method oralgorithm.

The circulating half-life of the peptidomimetic macrocycles in humanblood can be about 1-24 h. For example the circulating half-life of thepeptidomimetic macrocycles in human blood can me about 2-24 h, 4-24 h,6-24 h, 8-24 h, 10-24 h, 12-24 h, 14-24 h, 16-24 h, 18-24 h, 20-24 h,22-24 h, 1-20 h, 4-20 h, 6-20 h, 8-20 h, 10-20 h, 12-20 h, 14-20 h,16-20 h, 18-20 h, 1-16 b, 4-16 h, 6-16 h, 8-16 b, 10-16 b, 12-16 h,14-16 b, 1-12 h, 4-12 h, 6-12 h, 8-12 h, 10-12 h, 1-8 h, 4-8 h, 6-8 h,or 1-4 h. In some examples, the circulating half-life of thepeptidomimetic macrocycles in human blood can be bout 1-12 h, forexample about 1 h, 2 h, 3 h, 4 h, 5 h, 6 h, 7 h, 8 h, 9 h, 10 h, 11 h,or 12 h. In some examples, the circulating half-life of thepeptidomimetic macrocycles in human blood is about 2 h. In someexamples, the circulating half-life of the peptidomimetic macrocycles inhuman blood is about 4 h. In some examples, the circulating half-life ofthe peptidomimetic macrocycles in human blood is about 6 h. In someexamples, the circulating half-life of the peptidomimetic macrocycles inhuman blood is about 8 h. In some examples, the circulating half-life ofthe peptidomimetic macrocycles in human blood is about 10 h.

The half-life of the peptidomimetic macrocycles in biological tissue canbe about 1-24 h. For example the circulating half-life of thepeptidomimetic macrocycles in human blood can me about 1-24 h, 5-24 h,10-24 h, 15-24 h, 20-24 h, 1-22 h, 5-22 h, 10-22 h, 15-22 h, 20-22 h,1-20 h, 5-20 h, 15-20 h, 1-18 h, 5-18 h, 10-18 h, 15-18 h, 1-16 h, 5-16h, 10-16 h, 15-16 h, 1-14 h, 5-14 h, 10-14 h, 1-12 h, 5-12 h, 10-12 h,1-10 h, 5-10 h, 1-8 h, 5-8 h, 1-6 h, 5-6 h, or 1-4 h. In some examples,the circulating half-life of the peptidomimetic macrocycles in humanblood can be bout 5-20 h, for example about 5 h, 6 h, 7 h, 8 h, 9 h, 10h, 11 h, 12 h, 13 h, 14 h, 15 h, 16 h, 17 h, 18 h, 19 h or 20 h. In someexamples, the circulating half-life of the peptidomimetic macrocycles inhuman blood is about 2 h. In some examples, the circulating half-life ofthe peptidomimetic macrocycles in human blood is about 4 h. In someexamples, the circulating half-life of the peptidomimetic macrocycles inhuman blood is about 6 h. In some examples, the circulating half-life ofthe peptidomimetic macrocycles in human blood is about 8 h. In someexamples, the circulating half-life of the peptidomimetic macrocycles inhuman blood is about 10 h.

The circulating half-life of the peptidomimetic macrocycles in humanblood can be greater than, equal to, or less than the half-life of thepeptidomimetic macrocycles in biological tissue. In some examples, thecirculating half-life of the peptidomimetic macrocycles in human bloodcan be greater than the half-life of the peptidomimetic macrocycles inbiological tissue. In some examples, the circulating half-life of thepeptidomimetic macrocycles in human blood can be equal to the half-lifeof the peptidomimetic macrocycles in biological tissue. In someexamples, the half-life of the peptidomimetic macrocycles in biologicaltissue is greater than the circulating half-life of the peptidomimeticmacrocycles in human blood. This can facilitate administration of thepeptidomimetic macrocycles at a lower dose and/or at lower frequency. Insome embodiments, the half-life of the peptidomimetic macrocycles inbiological tissue is at least 1 h, 2 h, 3 h, 4 h, 5 h, 6 h, 7 h, 8 h, 9h, 10 h, 11 h, or 12 h greater than the than the circulating half-lifeof the peptidomimetic macrocycles in human blood. In some examples, thecirculating half-life of the peptidomimetic macrocycles in human bloodis about 4 h and the half-life of the in biological tissue is about 10h. In some examples, the circulating half-life of the peptidomimeticmacrocycles in human blood is about 6 h and the half-life of the inbiological tissue is about 10 h.

The cross-linked peptides of the disclosure can be modeled after theN-terminal transactivation domain of p53 (“p53 peptidomimeticmacrocycles”). These cross-linked peptides contain at least two modifiedamino acids that together form an intramolecular cross-link that canhelp to stabilize the α-helical secondary structure of a portion of p53that is thought to be important for binding of p53 to MDM2 and forbinding of p53 to MDMX. Accordingly, a cross-linked polypeptidedescribed herein can have improved biological activity relative to acorresponding polypeptide that is not cross-linked. The p53peptidomimetic macrocycles are thought to interfere with binding of p53to MDM2 and/or of p53 to MDMX, thereby liberating functional p53 andinhibiting its destruction. The p53 peptidomimetic macrocycles describedherein can be used therapeutically, for example to treat cancers andother disorders characterized by an undesirably low level or a lowactivity of p53, and/or to treat cancers and other disorderscharacterized by an undesirably high level of activity of MDM2 or MDMX.The p53 peptidomimetic macrocycles can also be useful for treatment ofany disorder associated with disrupted regulation of the p53transcriptional pathway, leading to conditions of excess cell survivaland proliferation such as cancer and autoimmunity, in addition toconditions of inappropriate cell cycle arrest and apoptosis such asneurodegeneration and immune deficiencies. In some embodiments, the p53peptidomimetic macrocycles bind to MDM2 (e.g., GenBank® Accession No.:228952: GI:228952) and/or MDMX (also referred to as MDM4; GenBank®Accession No.: 88702791; GI:88702791).

Table 1 shows a list of peptidomimetic macrocycles derived from theMDM2/MDMX-binding helix of p53 that were prepared. Tables 1a, 1b, 1c, 1dand 1e show a list of selected peptidomimetic macrocycles from Table 1.Table If shows a list of selected peptidomimetic macrocycles from Table1e. A partial staple scan was performed on the linear peptidep-CF₃-Phe7-D-PMI-β. SP-757, a potent and selective MDM2 antagonist, wasprepared by including an i, i+7 crosslink to the sequence ofp-CF3-Phe-7-D-PMI-b. SP-757 exhibited SJSA-1 sarcoma cell killingactivity at a single digit micromolar concentration (EC₅₀=1.5 mM). (FIG.3 ). SP-763 was prepared by increasing the alanine content to 35% whilemaintaining the net charge and Von Heijne score by adding four alanineresidues to the C-terminus of SP-757. SP-763 exhibited improved SJSA-1sarcoma cell killing activity (EC₅₀=0.15 mM) compared to SP-757 (FIG. 3). SP-763 exhibited similar cell killing activity as SP-449.

TABLE 1List of peptidomimetic macrocycles derived from the MDM2/MDMX-binding helix of p53 thatwere prepared. SEQ ID Exact Found Calc Calc Calc SP NO: Sequence IsomerMass Mass (M + 1)/1 (M + 2)/2 (M + 3)/3 SP1 9 Ac-F$r8AYWEAc3cL$AAA-NH21456.78 729.44 1457.79 729.4 486.6 SP2 10 Ac-F$r8AYWEAc3cL$AAibA-NH21470.79 736.4 1471.8 736.4 491.27 SP3 11 Ac-LTF$r8AYWAQL$SANle-NH21715.97 859.02 1716.98 858.99 573 SP4 12 Ac-LTF$r8AYWAQL$SAL-NH2 1715.97859.02 1716.98 858.99 573 SP5 13 Ac-LTF$r8AYWAQL$SAM-NH2 1733.92 868.481734.93 867.97 578.98 SP6 14 Ac-LTF$r8AYWAQL$SAhL-NH2 1729.98 865.981730.99 866 577.67 SP7 15 Ac-LTF$r8AYWAQL$SAF-NH2 1749.95 876.36 1750.96875.98 584.32 SP8 16 Ac-LTF$r8AYWAQL$SAI-NH2 1715.97 859.02 1716.98858.99 573 SP9 17 Ac-LTF$r8AYWAQL$SACHg-NH2 1741.98 871.98 1742.99 872581.67 SP10 18 Ac-LTF$r8AYWAQL$SAAib-NH2 1687.93 845.36 1688.94 844.97563.65 SP11 19 Ac-LTF$r8AYWAQL$SAA-NH2 1673.92 838.01 1674.93 837.97558.98 SP12 20 Ac-LTF$r8AYWASL$S$Nle-NH2 1767.04 884.77 1768.05 884.53590.02 SP13 21 Ac-LTF$r8AYWASL$S$A-NH2 1724.99 864.23 1726 863.5 576SP14 22 Ac-F$r8AYWEAc3cL$AANle-NH2 1498.82 750.46 1499.83 750.42 500.61SP15 23 Ac-F$r8AYWEAc3cL$AAL-NH2 1498.82 750.46 1499.83 750.42 500.61SP16 24 Ac-F$r8AYWEAc3cL$AAM-NH2 1516.78 759.41 1517.79 759.4 506.6 SP1725 Ac-F$r8AYWEAc3cL$AAhL-NH2 1512.84 757.49 1513.85 757.43 505.29 SP1826 Ac-F$r8AYWEAc3cL$AAF-NH2 1532.81 767.48 1533.82 767.41 511.94 SP19 27Ac-F$r8AYWEAc3cL$AAI-NH2 1498.82 750.39 1499.83 750.42 500.61 SP20 28Ac-F$r8AYWEAc3cL$AAChg-NH2 1524.84 763.48 1525.85 763.43 509.29 SP21 29Ac-F$r8AYWEAc3cL$AACha-NH2 1538.85 770.44 1539.86 770.43 513.96 SP22 30Ac-F$r8AYWEAc3cL$AAAib-NH2 1470.79 736.84 1471.8 736.4 491.27 SP23 31Ac-LTF$r8AYWAQL$AAAibV-NH2 1771.01 885.81 1772.02 886.51 591.34 SP24 32Ac-LTF$r8AYWAQL$AAAibV-NH2 iso2 1771.01 886.26 1772.02 886.51 591.34SP25 33 Ac-LTF$r8AYWAQL$SAibAA-NH2 1758.97 879.89 1759.98 880.49 587.33SP26 34 Ac-LTF$r8AYWAQL$SAibAA-NH2 iso2 1758.97 880.34 1759.98 880.49587.33 SP27 35 Ac-HLTF$r8HHWHQL$AANleNle- 2056.15 1028.86 2057.161029.08 686.39 NH2 SP28 36 Ac-DLTF$r8HHWHQL$RRLV-NH2 2190.23 731.152191.24 1096.12 731.08 SP29 37 Ac-HHTF$r8HHWHQL$AAML-NH2 2098.08 700.432099.09 1050.05 700.37 SP30 38 Ac-F$r8HHWHQL$RRDCha-NH2 1917.06 959.961918.07 959.54 640.03 SP31 39 Ac-F$r8HHWHQL$HRFV-NH2 1876.02 938.651877.03 939.02 626.35 SP32 40 Ac-HLTF$r8HHWHQL$AAhLA-NH2 2028.12 677.22029.13 1015.07 677.05 SP33 41 Ac-DLTF$r8HHWHQL$RRCHgl-NH2 2230.261115.89 2231.27 1116.14 744.43 SP34 42 Ac-DLTF$r8HHWHQL$RRCHgl-NH2 iso22230.26 1115.96 2231.27 1116.14 744.43 SP35 43Ac-HHTF$r8HHWHQL$AAChav-NH2 2106.14 1053.95 2107.15 1054.08 703.05 SP3644 Ac-F$rF8HHWHQL$RRDa-NH2 1834.99 918.3 1836 918.5 612.67 SP37 45Ac-F$r8HHWHQL$HRAibG-NH2 1771.95 886.77 1772.96 886.98 591.66 SP38 46Ac-F$r8AYWAQL$HHNleL-NH2 1730.97 866.57 1731.98 866.49 578 SP39 47Ac-F$r8AYWSAL$HQANle-NH2 1638.89 820.54 1639.9 820.45 547.3 SP40 48Ac-F$r8AYWVQL$QHChgl-NH2 1776.01 889.44 1777.02 889.01 593.01 SP41 49Ac-F$r8AYWTAL$QQNlev-NH2 1671.94 836.97 1672.95 836.98 558.32 SP42 50Ac-F$r8AYWYQL$HAibAa-NH2 1686.89 844.52 1687.9 844.45 563.3 SP43 51Ac-LTF$r8AYWAQL$HHLa-NH2 1903.05 952.27 1904.06 952.53 635.36 SP44 52Ac-LTF$r8AYWAQL$HHLa-NH2 iso2 1903.05 952.27 1904.06 952.53 635.36 SP4553 Ac-LTF$r8AYWAQL$HQNlev-NH2 1922.08 962.48 1923.09 962.05 641.7 SP4654 Ac-LTF$r8AYWAQL$HQNlev-NH2 iso2 1922.08 962.4 1923.09 962.05 641.7SP47 55 Ac-LTF$r8AYWAQL$QQMl-NH2 1945.05 973.95 1946.06 973.53 649.36SP48 56 Ac-LTF$r8AYWAQL$QQMl-NH2 iso2 1945.05 973.88 1946.06 973.53649.36 SP49 57 Ac-LTF$r8AYWAQL$HAibhLV-NH2 1893.09 948.31 1894.1 947.55632.04 SP50 58 Ac-LTF$r8AYWAQL$AHFA-NH2 1871.01 937.4 1872.02 936.51624.68 SP51 59 Ac-HLTF$r8HHWHQL$AANlel-NH2 2056.15 1028.79 2057.161029.08 686.39 SP52 60 Ac-DLTF$r8HHWHQL$RRLa-NH2 2162.2 721.82 2163.211082.11 721.74 SP53 61 Ac-HHTF$r8HHWHQL$AAMv-NH2 2084.07 1042.92 2085.081043.04 695.7 SP54 62 Ac-F$r8HHWHQL$RRDA-NH2 1834.99 612.74 1836 918.5612.67 SP55 63 Ac-F$r8HHWHQL$HRFCha-NH2 1930.06 966.47 1931.07 966.04644.36 SP56 64 Ac-F$r8AYWEAL$AA-NHAm 1443.82 1445.71 1444.83 722.92482.28 SP57 65 Ac-F$r8AYWEAL$AA-NHiAm 1443.82 723.13 1444.83 722.92482.28 SP58 66 Ac-F$r8AYWEAL$AA-NHnPr3Ph 1491.82 747.3 1492.83 746.92498.28 SP59 67 Ac-F$r8AYWEAL$AA-NHnBu33Me 1457.83 1458.94 1458.84 720 92486.95 SP60 68 Ac-F$r8AYWEAL$AA-NHnPr 1415.79 709.28 1416.8 708.9 472.94SP61 69 Ac-F$r8AYWEAL$AA-NHnEt2Ch 1483.85 1485.77 1484.86 742.93 495.62SP62 70 Ac-F$r8AYWEAL$AA-NHnEt2Cp 1469.83 1470.78 1470.84 735.92 490.95SP63 71 Ac-F$r8AYWEAL$AA-NHHex 1457.83 730.19 1458.84 729.92 486.95 SP6472 Ac-LTF$r8AYWAQL$AAIA-NH2 1771.01 885.81 1772.02 886.51 591.34 SP65 73Ac-LTF$r8AYWAQL$AAIA-NH2 iso2 1771.01 866.8 1772.02 886.51 591.34 SP6674 Ac-LTF$r8AYWAAL$AAMA-NH2 1731.94 867.08 1732.95 866.98 578.32 SP67 75Ac-LTF$r8AYWAAL$AAMA-NH2 iso2 1731.94 867.28 1732.95 866.98 578.32 SP6876 Ac-LTF$r8AYWAQL$AANleA-NH2 1771.01 867.1 1772.02 886.51 591.34 SP6977 AC-LTF$r8AYWAQL$AANleA-NH2 iso2 1771.01 886.89 1772.02 886.51 591.34SP70 78 Ac-LTF$r8AYWAQL$AAIa-NH2 1771.01 886.8 1772.02 886.51 591.34SP71 79 Ac-LTF$r8AYWAQL$AAIa-NH2 iso2 1771.01 887.09 1772.02 886.51591.34 SP72 80 Ac-LTF$r8AYWAAL$AAMa-NH2 1731.94 867.17 1732.95 866.98578.32 SP73 81 Ac-LTF$r8AYWAAL$AAMa-NH2 iso2 1731.94 867.37 1732.95866.98 578.32 SP74 82 Ac-LTF$r8AYWAQL$AANlea-NH2 1771.01 887.08 1772.02886.51 591.34 SP75 83 Ac-LTF$r8AYWAQL$AANlea-NH2 iso2 1771.01 887.081772.02 886.51 591.34 SP76 84 Ac-LTF$r8AYWAAL$AAIv-NH2 1742.02 872.371743.03 872.02 581.68 SP77 85 Ac-LTF$r8AYWAAL$AAIv-NH2 iso2 1742.02872.74 1743.03 872.02 581.68 SP78 86 Ac-LTF$r8AYWAQL$AAMv-NH2 1817910.02 1818.01 909.51 606.67 SP79 87 Ac-LTF$r8AYWAAL$AANlev-NH2 1742.02872.37 1743.03 872.02 581.68 SP80 88 Ac-LTF$r8AYWAAL$AANlev-NH2 iso21742.02 872.28 1743.03 872.02 581.68 SP81 89 Ac-LTF$r8AYWAQL$AAIl-NH21813.05 907.81 1814.06 907.53 605.36 SP82 90 Ac-LTF$r8AYWAQL$AAIl-NH2iso2 1813.05 907.81 1814.06 907.53 605.36 SP83 91Ac-LTF$r8AYWAAL$AAMl-NH2 1773.99 887.37 1775 888 592.34 SP84 92Ac-LTF$r8AYWAQL$AANlel-NH2 1813.05 907.61 1814.06 907.53 605.36 SP85 93Ac-LTF$r8AYWAQL$AANlel-NH2 iso2 1813.05 907.71 1814.06 907.53 605.36SP86 94 Ac-F$r8AYWEAL$AAMA-NH2 1575.82 789.02 1576.83 788.92 526.28 SP8795 Ac-F$r8AYWEAL$AANleA-NH2 1557.86 780.14 1558.87 779.94 520.29 SP88 96Ac-F$r8AYWEAL$AAIa-NH2 1557.86 780.33 1558.87 779.94 520.29 SP89 97Ac-F$r8AYWEAL$AAMa-NH2 1575.82 789.3 1576.83 788.92 526.28 SP90 98Ac-F$r8AYWEAL$AANlea-NH2 1557.86 779.4 1558.87 779.94 520.29 SP91 99Ac-F$r8AYWEAL$AAIv-NH2 1585.89 794.29 1586.9 793.95 529.64 SP92 100Ac-F$r8AYWEAL$AAMv-NH2 1603.85 803.08 1604.86 802.93 535.62 SP93 101Ac-F$r8AYWEAL$AANlev-NH2 1585.89 793.46 1586.9 793.95 529.64 SP94 102Ac-F$r8AYWEAL$AAIl-NH2 1599.91 800.49 1600.92 800.96 534.31 SP95 103Ac-F$r8AYWEAL$AAMl-NH2 1617.86 809.44 1618.87 809.94 540.29 SP96 104Ac-F$r8AYWEAL$AANlel-NH2 1599.91 801.7 1600.92 800.96 534.31 SP97 105Ac-F$r8AYWEAL$AANlel-NH2 iso2 1599.91 801.42 1600.92 800.96 534.31 SP98106 Ac-LTF$r8AYSclWAQL$SAA-NH2 1707.88 855.72 1708.89 854.95 570.3 SP99107 Ac-LTF$r8AY6clWAQL$SAA-NH2 iso2 1707.88 855.35 1708.89 854.95 570.3SP100 108 Ac-WTF$r8FYWSQL$AVAa-NH2 1922.01 962.21 1923.02 962.01 641.68SP101 109 Ac-WTF$r8FYWSQL$AVAa-NH2 iso2 1922.01 962.49 1923.02 962.01641.68 SP102 110 Ac-WTF$r8VYWSQL$AVA -NH2 1802.98 902.72 1803.99 902.5602 SP103 111 Ac-WTF$r8VYWSQL$AVA-NH2 iso2 1802.98 903 1803.99 902.5 602SP104 112 Ac-WTF$r8FYWSQL$SAAa-NH2 1909.98 956.47 1910.99 956 637.67SP105 113 Ac-WTF$r8FYWSQL$SAAa-NH2 iso2 1909.98 956.47 1910.99 956637.67 SP106 114 Ac-WTF$r8VYWSQL$AVAaa-NH2 1945.05 974.15 1946.06 973.53649.36 SP107 115 Ac-WTF$r8VYWSQL$AVAaa-NH2 iso2 1945.05 973.78 1946.06973.53 649.36 SP108 116 Ac-LTF$r8AYWAQL$AVG-NH2 1671.94 837.52 1672.95836.98 558.32 SP109 117 Ac-LTF$r8AYWAQL$AVG-NH2 iso2 1671.94 837.211672.95 836.98 558.32 SP110 118 Ac-LTF$r8AYWAQL$AVQ-NH2 1742.98 872.741743.99 872.5 582 SP1I1 119 Ac-LTF$r8AYWAQL$AVQ-NH2 iso2 1742.98 872.741743.99 872.5 582 SP112 120 Ac-LTF$r8AYWAQL$SAa-NH2 1673.92 838.231674.93 837.97 558.98 SP113 121 Ac-LTF$r8AYWAQL$SAa-NH2 iso2 1673.92838.32 1674.93 837.97 558.98 SP114 122 Ac-LTF$r8AYWAQhL$SAA-NH2 1687.93844.37 1688.94 844.97 563.65 SP115 123 Ac-LTF$r8AYWAQhL$SAA-NH2 iso21687.93 844.81 1688.94 844.97 563.65 SP116 124 Ac-LTF$r8AYWEQLStSA#-NH21826 905.27 1827.01 914.01 609.67 SP117 125 Ac-LTF$r8AYWAQL$SLA-NH21715.97 858.48 1716.98 858.99 573 SP118 126 Ac-LTF$r8AYWAQL$SLA-NH2 iso21715.97 858.87 1716.98 858.99 573 SP119 127 Ac-LTF$r8AYWAQL$SWA-NH21788.96 895.21 1789.97 895.49 597.33 SP120 128 Ac-LTF$r8AYWAQL$SWA-NH2iso2 1788.96 895.28 1789.97 895.49 597.33 SP121 129Ac-LTF$r8AYWAQL$SVS-NH2 1717.94 859.84 1718.95 859.98 573.65 SP122 130Ac-LTF$r8AYWAQL$SAS-NH2 1689.91 845.85 1690.92 845.96 564.31 SP123 131Ac-LTF$r8AYWAQL$SVG-NH2 1687.93 844.81 1688.94 844.97 563.65 SP124 132Ac-ETF$r8VYWAQL$SAa-NH2 1717.91 859.76 1718.92 859.96 573.64 SP125 133Ac-ETF$r8VYWAQL$SAA-NH2 1717.91 859.84 1718.92 859.96 573.64 SP126 134Ac-ETF$r8VYWAQL$SVA-NH2 1745.94 873.82 1746.95 873.98 582.99 SP127 135Ac-ETF$r8VYWAQL$SLA-NH2 1759.96 880.85 1760.97 880.99 587.66 SP128 136Ac-ETF$r8VYWAQL$SWA-NH2 1832.95 917.34 1833.96 917.48 611.99 SP129 137Ac-ETF$r8KYWAQL$SWA-NH2 1861.98 931.92 1862.99 932 621.67 SP130 138Ac-ETF$r8VYWAQL$SVS-NH2 1761.93 881.89 1762.94 881.97 588.32 SP131 139Ac-ETF$r8VYWAQL$SAS-NH2 1733.9 867.83 1734.91 867.96 578.97 SP132 140Ac-ETF$r8VYWAQL$SVG-NH2 1731.92 866.87 1732.93 866.97 578.31 SP133 141Ac-LTF$r8VYWAQL$SSa-NH2 1717.94 859.47 1718.95 859.98 573.65 SP134 142Ac-ETF$r8VYWAQL$SSa-NH2 1733.9 867.83 1734.91 867.96 578.97 SP135 143Ac-LTF$r8VYWAQL$SNa-NH2 1744.96 873.38 1745.97 873.49 582.66 SP136 144Ac-ETF$r8VYWAQL$SNa-NH2 1760.91 881.3 1761.92 881.46 587.98 SP137 145Ac-LTF$r8VYWAQL$SAa-NH2 1701.95 851.84 1702.96 851.98 568.32 SP138 146Ac-LTF$r8VYWAQL$SVA-NH2 1729.98 865.53 1730.99 866 577.67 SP139 147Ac-LTF$r8VYWAQL$SVA-NH2 iso2 1729.98 865.9 1730.99 866 577.67 SP140 148Ac-LTF$r8VYWAQL$SWA-NH2 1816.99 909.42 1818 909.5 606.67 SP141 149Ac-LTF$r8VYWAQL$SVS-NH2 1745.98 873.9 1746.99 874 583 SP142 150Ac-LTF$r8VYWAQL$SVS-NH2 iso2 1745.98 873.9 1746.99 874 583 SP143 151Ac-LTF$r8VYWAQL$SAS-NH2 1717.94 859.84 1718.95 859.98 573.65 SP144 152Ac-LTF$r8VYWAQL$SAS-NH2 iso2 1717.94 859.91 1718.95 859.98 573.65 SP145153 Ac-LTF$r8VYWAQL$SVG-NH2 1715.97 858.87 1716.98 858.99 573 SP146 154Ac-LTF$r8VYWAQL$SVG-NH2 iso2 1715.97 858.87 1716.98 858.99 573 SP147 155Ac-LTF$r8EYWAQCha$SAA-NH2 1771.96 886.85 1772.97 886.99 591.66 SP148 156Ac-LTF$r8EYWAQCha$SAA-NH2 iso2 1771.96 886.85 1772.97 886.99 591.66SP149 157 Ac-LTF$r8EYWAQCpg$SAA-NH2 1743.92 872.86 1744.93 872.97 582.31SP150 158 Ac-LTF$r8EYWAQCPg$SAA-NH2 iso2 1743.92 872.86 1744.93 872.97582.31 SP151 159 Ac-LTF$r8EYWAQF$SAA-NH2 1765.91 883.44 1766.92 883.96589.64 SP152 160 Ac-LTF$r8EYWAQF$SAA-NH2 iso2 1765.91 883.89 1766.92883.96 589.64 SP153 161 Ac-LTF$r8EYWAQCba$SAA-NH2 1743.92 872.42 1744.93872.97 582.31 SP154 162 Ac-LTF$r8EYWAQCba$SAA-NH2 iso2 1743.92 873.391744 93 872.97 582.31 SP155 163 Ac-LTF3Cl$r8EYWAQL$SAA-NH2 1765.89883.89 1766.9 883.95 589.64 SP156 164 Ac-LTF3Cl$r8EYWAQL$SAA-NH2 iso21765.89 883.96 1766.9 883.95 589.64 SP157 165Ac-LTF34F2$r8EYWAQL$SAA-NH2 1767.91 884.48 1768.92 884.96 590.31 SP158166 Ac-LTF34F2$r8EYWAQL$SAA-NH2 iso2 1767.91 884.48 1768.92 884.96590.31 SP159 167 Ac-LTF34F2$r8EYWAQhL$SAA- 1781.92 891.44 1782.93 891.97594.98 NH2 SP160 168 Ac-LTF34F2$r8EYWAQhL$SAA- iso2 1781.92 891.881782.93 891.97 594.98 NH2 SP161 169 Ac-ETF$r8EYWAQL$SAA-NH2 1747.88874.34 1748.89 874.95 583.63 SP162 170 Ac-LTF$r8AYWVQL$SAA-NH2 1701.95851.4 1702.96 851.98 568.32 SP163 171 Ac-LTF$8AHWAQL$SAA-NH2 1647.91824.83 1648.92 824.96 550.31 SP164 172 Ac-LTF$r8AEWAQL$SAA-NH2 1639.9820.39 1640.91 820.96 547.64 SP165 173 Ac-LTF$r8ASWAQL$SAA-NH2 1597.89799.38 1598.9 799.95 533.64 SP166 174 Ac-LTF$r8AEWAQL$SAA-NH2 iso21639.9 820.39 1640.91 820.96 547.64 SP167 175 Ac-LTF$r8ASWAQL$SAA-NH2iso2 1597.89 800.31 1598.9 799.95 533.64 SP168 176Ac-LTF$r8AF4coohWAQL$SAA-NH2 1701.91 851.4 1702.92 851.96 568.31 SP169177 Ac-LTF$r8AF4coohWAQL$SAA-NH2 iso2 1701.91 851.4 1702.92 851.96568.31 SP170 178 Ac-LTF$r8AHWAQL$AAIa-NH2 1745 874.13 1746.01 873.51582.67 SP171 179 Ac-ITF$r8FYWAQL$AAIa-NH2 1847.04 923.92 1848.05 924.53616.69 SP172 180 Ac-ITF$r8EHWAQL$AAIa-NH2 1803.01 903.17 1804.02 902.51602.01 SP173 181 Ac-ITF$rEHWAQL$AAIa-NH2 iso2 1803.01 903.17 1804.02902.51 602.01 SP174 182 Ac-ETF$r8EHWAQL$AAIa-NH2 1818.97 910.76 1819.98910.49 607.33 SP175 183 Ac-ETF$r8EHWAQL$AAIa-NH2 iso2 1818.97 910.851819.98 910.49 607.33 SP176 184 Ac-LTF$r8AHWVQL$AAIa-NH2 1773.03 888.091774.04 887.52 592.02 SP177 185 Ac-ITF$r8FYWVQL$AAIa-NH2 1875.07 939.161876.08 938.54 626.03 SP178 186 Ac-ITF$r8EYWVQL$AAIa-NH2 1857.04 929.831858.05 929.53 620.02 SP179 187 Ac-ITF$r8EHWVQL$AAIa-NH2 1831.04 916.861832.05 916.53 611.35 SP180 188 Ac-LTF$r8AEWAQL$AAIa-NH2 1736.99 869.871738 869.5 580 SP181 189 Ac-LTF$r8AF4coohWAQL$AAIa- 1799 900.17 1800.01900.51 600.67 NH2 SP182 190 Ac-LTF$r8AF4coohWAQL$AAIa- iso2 1799 900.241800.01 900.51 600.67 NH2 SP183 191 Ac-LTF$r8AHWAQL$AHFA-NH2 1845.01923.89 1846.02 923.51 616.01 SP184 192 Ac-ITF$r8FYWAQL$AHFA-NH2 1947.05975.05 1948.06 974.53 650.02 SP1 85 193 Ac-ITF$r8HYWAQL$AHFA-NH2 iso21947.05 976.07 1948.06 974.53 650.02 SP186 194 Ac-ITF$r8FHWAQL$AEFA-NH21913.02 958.12 1914.03 957.52 638.68 SP187 195 Ac-ITF$r8FHWAQL$AEFA-NH2iso2 1913.02 957.86 1914.03 957.52 638.68 SP188 196Ac-ITF$r8EHWAQL$AHFA-NH2 1903.01 952.94 1904.02 952.51 635.34 SP189 197Ac-ITF$r8EHWAQL$AHFA-NH2 iso2 1903.01 953.87 1904.02 952.51 635.34 SP190198 Ac-LTF$r8AHWVQL$AHFA-NH2 1873.04 937.86 1874.05 937.53 625.35 SP191199 Ac-ITF$r8FYWVQL$AHFA-NH2 1975.08 988.83 1976.09 988.55 659.37 SP192200 Ac-ITF$r8EYWVQL$AHFA-NH2 1957.05 979.35 1958.06 979.53 653.36 SP193201 AC-ITF$r8EHWVQL$AHFA-NH2 1931.05 967 1932.06 966.53 644.69 SP194 202Ac-ITF$r8EHWVQL$AHFA-NH2 iso2 1931.05 967.93 1932.06 966.53 644.69 SP195203 Ac-ETF$r8EYWAAL$SAA-NH2 1690.86 845.85 1691.87 846.44 564.63 SP196204 Ac-LTF$r8AYWVAL$SAA-NH2 1644.93 824.08 1645.94 823.47 549.32 SP197205 Ac-LTF$r8AHWAAL$SAA-NH2 1590.89 796.88 1591.9 796.45 531.3 SP198 206Ac-LTF$r8AEWAAL$SAA-NH2 1582.88 791.9 1583.89 792.45 528.63 SP199 207Ac-LTF$r8AEWAAL$SAA-NH2 iso2 1582.88 791.9 1583.89 792.45 528.63 SP200208 Ac-LTF$r8ASWAAL$SAA-NH2 1540.87 770.74 1541.88 771.44 514.63 SP201209 Ac-LTF$r8ASWAAL$SAA-NH2 iso2 1540.87 770.88 1541.88 771.44 514.63SP202 210 Ac-LTF$r8AYWAAL$AAIa-NH2 1713.99 857.39 1715 858 572.34 SP203211 Ac-LTF$r8AYWAAL$AAIa-NH2 iso2 1713.99 857.84 1715 858 572.34 SP204212 Ac-LTF$r8AYWAAL$AHFA-NH2 1813.99 907.86 1815 908 605.67 SP205 213Ac-LTF$r8EHWAQL$AHIa-NH2 1869.03 936.1 1870.04 935.52 624.02 SP206 214Ac-LTF$r8EHWAQL$AHIa-NH2 iso2 1869.03 937.03 1870.04 935.52 624.02 SP207215 Ac-LTF$r8AHWAQL$AHIa-NH2 1811.03 906.87 1812.04 906.52 604.68 SP208216 Ac-LTF$r8EYWAQL$AHIa-NH2 1895.04 949.15 1896.05 948.53 632.69 SP209217 Ac-LTF$r8AYWAQL$AAFa-NH2 1804.99 903.2 1806 903.5 602.67 SP210 218Ac-LTF$r8AYWAQL$AAFa-NH2 iso2 1804.99 903.28 1806 903.5 602.67 SP211 219Ac-LTF$r8AYWAQL$AAWa-NH2 1844 922.81 1845.01 923.01 615.67 SP212 220Ac-LTF$r8AYWAQL$AAVa-NH2 1756.99 878.86 1758 879.5 586.67 SP213 221Ac-LTF$r8AYWAQL$AAVa-NH2 iso2 1756.99 879.3 1758 879.5 586.67 SP214 222Ac-LTF$r8AYWAQL$AALa-NH2 1771.01 886.26 1772.02 886.51 591.34 SP215 223Ac-LTF$r8AYWAQL$AALa-NH2 iso2 1771.01 886.33 1772.02 886.51 591.34 SP216224 Ac-LTF$r8EYWAQL$AAIa-NH2 1829.01 914.89 1830.02 915.51 610.68 SP217225 Ac-LTF$r8EYWAQL$AAIa-NH2 iso2 1829.01 915.34 1830.02 915.51 610.68SP218 226 Ac-LTF$r8EYWAQL$AAFa-NH2 1863 932.87 1864.01 932.51 622.01SP219 227 Ac-LTF$r8EYWAQL$AAFa-NH2 iso2 1863 932.87 1864.01 932.51622.01 SP220 228 Ac-LTF$r8EYWAQL$AAVa-NH2 1815 908.23 1816.01 908.51606.01 SP221 229 Ac-LTF$r8EYWAQL$AAVa-NH2 iso2 1815 908.31 1816.01908.51 606.01 SP222 230 Ac-LTF$r8EHWAQL$AAIa-NH2 1803.01 903.17 1804.02902.51 602.01 SP223 231 Ac-LTF$r8EHWAQL$AAIa-NH2 iso2 1803.01 902.81804.02 902.51 602.01 SP224 232 Ac-LTF$r8EHWAQL$AAWa-NH2 1876 939.341877.01 939.01 626.34 SP225 233 Ac-LTF$r8EHWAQL$AAWa-NH2 iso2 1876939.62 1877.01 939.01 626.34 SP226 234 Ac-LTF$r8EHWAQL$AALa-NH2 1803.01902.8 1804.02 902.51 602.01 SP227 235 Ac-LTF$r8EHWAQL$AALa-NH2 iso21803.01 902.9 1804.02 902.51 602.01 SP228 236 Ac-ETF$r8EHWVQL$AALa-NH21847 924.82 1848.01 924.51 616.67 SP229 237 Ac-LTF$r8AYWAQL$AAAa-NH21728.96 865.89 1729.97 865.49 577.33 SP230 238 Ac-LTF$r8AYWAQL$AAAa-NH2iso2 1728.96 865.89 1729.97 865.49 577.33 SP231 239Ac-LTF$r8AYWAQL$AAAibA-NH2 1742.98 872.83 1743.99 872.5 582 SP232 240Ac-LTF$r8AYWAQL$AAAibA-NH2 iso2 1742.98 872.92 1743.99 872.5 582 SP233241 Ac-LTF$r8AYWAQL$AAAAa-NH2 1800 901.42 1801.01 901.01 601.01 SP234242 Ac-LTF$r8AYWAQL$s8AAIa-NH2 1771.01 887.17 1772.02 886.51 591.34SP235 243 Ac-LTF$r8AYWAQL$s8SAA-NH2 1673.92 838.33 1674.93 837.97 558.98SP236 244 Ac-LTF$r8AYWAQCba$AANleA- 1783.01 892.64 1784.02 892.51 595.34NH2 SP237 245 Ac-ETF$r8AYWAQCba$AANleA- 1798.97 900.59 1799.98 900.49600.66 NH2 SP238 246 Ac-LTF$r8EYWAQCba$AANleA- 1841.01 922.05 1842.02921.51 614.68 NH2 SP239 247 Ac-LTF$r8AYWAQCba$AWNleA- 1898.05 950.461899.06 950.03 633.69 NH2 SP240 248 Ac-ETF$r8AYWAQCba$AWNleA- 1914.01958.11 1915.02 958.01 639.01 NH2 SP241 249 Ac-LTF$r8EYWAQCba$AWNleA-1956.06 950.62 1957.07 979.04 653.03 NH2 SP242 250Ac-LTF$r8EYWAQCba$SAFA-NH2 1890.99 946.55 1892 946.5 631.34 SP243 251Ac- 1892.99 947.57 1894 947.5 632 LTF34F2$r8EYWAQCba$SANleA- NH2 SP244252 Ac- 1885 943.59 1886.01 943.51 629.34 LTF$r8EF4coohWAQCba$SANleA-NH2 SP245 253 Ac-LTF$r8EYWSQCba$SANleA-NH2 1873 937.58 1874.01 937.51625.34 SP246 254 Ac-LTF$r8EYWWQCba$SANleA- 1972.05 987.61 1973.06 987.03658.36 NH2 SP247 255 Ac-LTF$r8EYWAQCba$AAIa-NH2 1841.01 922.05 1842.02921.51 614.68 SP248 256 Ac-LTF34F2$r8EYWAQCba$SAAla- 1876.99 939.99 1878939.5 626.67 NH2 SP249 257 Ac-LTF$r8EF4coohWAQCba$AAIa- 1869.01 935.641870.02 935.51 624.01 NH2 SP250 258 Pam-ETF$r8EYWAQCba$SAA-NH2 1956.1979.57 1957.11 979.06 653.04 SP251 259 Ac-LThF$r8EFWAQCba$SAA-NH21741.94 872.11 1742.95 871.98 581.65 SP252 260 Ac-LTA$r8EYWAQCba$SAA-NH21667.89 835.4 1668.9 834.95 556.97 SP253 261 Ac-LTF$r8EYAAQCba$SAA-NH21628.88 815.61 1629.89 815.45 543.97 SP254 262Ac-LTF$r8EY2NalAQCba$SAA-NH2 1754.93 879.04 1755.94 878.47 585.98 SP255263 Ac-LTF$r8AYWAQCba$SAA-NH2 1685.92 844.71 1686.93 843.97 562.98 SP256264 Ac-LTF$r8EYWAQCba$SAF-NH2 1819.96 911.41 1820.97 910.99 607.66 SP257265 Ac-LTF$r8EYWAQCba$SAFa-NH2 1890.99 947.41 1892 946.5 631.34 SP258266 Ac-LTF$r8AYWAQCba$SAF-NH2 1761.95 882.73 1762.96 881.98 588.32 SP259267 Ac-LTF34F2$r8AYWAQCba$SAF- 1797.93 900.87 1798.94 899.97 600.32 NH2SP260 268 Ac-LTF$r8AF4coohWAQCba$SAF- 1789.94 896.43 1790.95 895.98597.65 NH2 SP261 269 Ac-LTF$r8EY6clWAQCba$SAF-NH2 1853.92 929.27 1854.93927.97 618.98 SP262 270 Ac-LTF$r8AYWSQCba$SAF-NH2 1777.94 890.87 1778.95889.98 593.65 SP263 271 Ac-LTF$r8AYWWQCba$SAF-NH2 1876.99 939.91 1878939.5 626.67 SP264 272 Ac-LTF$r8AYWAQCba$AAIa-NH2 1783.01 893.19 1784.02892.51 595.34 SP265 273 Ac-LTF34F2$r8AYWAQCba$AAIa- 1818.99 911.23 1820910.5 607.34 NH2 SP266 274 Ac-LTF$r8AY6clWAQCba$AAIa- 1816.97 909.841817.98 909.49 606.66 NH2 SP267 275 Ac-LTF$r8AF4coohWAQCbaRAAIa- 1811906.88 1812.01 906.51 604.67 NH2 SP268 276 Ac-LTF$r8EYWAQCba$AAFa-NH21875 938.6 1876.01 938.51 626.01 SP269 277 Ac-LTF$r8EYWAQCba$AAFa-NH2iso2 1875 938.6 1876.01 938.51 626.01 SP270 278Ac-ETF$r8AYWAQCba$AWNlea- 1914.01 958.42 1915.02 958.01 639.01 NH2 SP271279 Ac-LTF$r8EYWAQCba$AWNlea- 1956.06 979.42 1957.07 979.04 653.03 NH2SP272 280 Ac-ETF$r8EYWAQCba$AWNlea- 1972.01 987.06 1973.02 987.01 658.34NH2 SP273 281 Ac-ETF$r8EYWAQCba$AWNlea- iso2 1972.01 987.06 1973.02987.01 658.34 NH2 SP274 282 Ac-LTF$r8AYWAQCba$SAFa-NH2 1832.99 917.891834 917.5 612 SP275 283 Ac-LTF$r8AYWAQCba$SAFa-NH2 iso2 1832.99 918.071834 917.5 612 SP276 284 Ac-ETF$r8AYWAQL$AWNlea-NH2 1902.01 952.221903.02 952.01 635.01 SP277 285 Ac-LTF$r8EYWAQL$AWNlea-NH2 1944.06 973.51945.07 973.04 649.03 SP278 286 Ac-ETF$r8EYWAQL$AWNlea-NH2 1960.01981.46 1961.02 981.01 654.34 SP279 287 Dmaac-LTF$r8EYWAQhL$SAA-NH21788.98 896.06 1789.99 895.5 597.33 SP280 288Hexac-LTF$r8EYWAQhL$SAA-NH2 1802 902.9 1803.01 902.01 601.67 SP281 289Napac-LTF$r8EYWAQhL$SAA-NH2 1871.99 937.58 1873 937 625 SP282 290Decac-LTF$r8EYWAQhL$SAA-NH2 1858.06 930.55 1859.07 930.04 620.36 SP283291 Admac-LTF$r8EYWAQhL$SAA-NH2 1866.03 934.07 1867.04 934.02 623.02SP284 292 Tmac-LTF$r8EYWAQhL$SAA-NH2 1787.99 895.41 1789 895 597 SP285293 Pam-LTF$r8EYWAQhL$SAA-NH2 1942.16 972.08 1943.17 972.09 648.39 SP286294 Ac-LTF$r8AYWAQCba$AANleA- iso2 1783.01 892.64 1784.02 892.51 595.34NH2 SP287 295 Ac-LTF34F2$Sr8EYWAQCba$AAIa- iso2 1876.99 939.62 1878939.5 626.67 NH2 SP288 296 Ac-LTF34F2Rr8EYWAQCba$SAA- 1779.91 892.071780.92 890.96 594.31 NH2 SP289 297 Ac-LTF34F2$r8EYWAQCba$SAA- iso21779.91 891.61 1780.92 890.96 594.31 NH2 SP290 298Ac-LTF$r8EF4coohWAQCba$SAA- 1771.92 887.54 1772.93 886.97 591.65 NH2SP291 299 Ac-LTF$r8EF4coohWAQCba$SAA- iso2 1771.92 887.63 1772.93 886.97591.65 NH2 SP292 300 Ac-LTF$r8EYWSQCba$SAA-NH2 1759.92 881.9 1760.93880.97 587.65 SP293 301 Ac-LTF$r8EYWSQCba$SAA-NH2 iso2 1759.92 881.91760.93 880.97 587.65 SP294 302 Ac-LTF$r8EYWAQhL$SAA-NH2 1745.94 875.051746.95 873.98 582.99 SP295 303 Ac-LlT$r8AYWAQhL$SAF-NH2 1763.97 884.021764.98 882.99 589 SP296 304 Ac-LTF$r8AYWAQhL$SAF-NH2 iso2 1763.97883.56 1764.98 882.99 589 SP297 305 Ac-LTF34F2$r8AYWAQhL$SAA- 1723.92863.67 1724.93 862.97 575.65 NH2 SP298 306 Ac-LTF34F2$r8AYWAQhL$SAA-iso2 1723.92 864.04 1724.93 862.97 575.65 NH2 SP299 307Ac-LTF$r8AF4coohWAQhL$SAA- 1715.93 859.44 1716.94 858.97 572.98 NH2SP300 308 Ac-LTF$r8AF4coohWAQhL$SAA- iso2 1715.93 859.6 1716.94 858.97572.98 NH2 SP301 309 Ac-LTF$r8AYWSQhl$SAA-NH2 1703.93 853.96 1704.94852.97 568.98 SP302 310 Ac-LTF$r8AYWSQhL$SAA-NH2 iso2 1703.93 853.591704.94 852.97 568.98 SP303 311 Ac-LTF$r8EYWAQL$AANleA-NH2 1829.01915.45 1830.02 915.51 610.68 SP304 312 Ac-LTF34F2$r8AYWAQL$AANleA-1806.99 904.58 1808 904.5 603.34 NH2 SP305 313Ac-LTF$r8AF4coohWAQL$AANleA- 1799 901.6 1800.01 900.51 600.67 NH2 SP306314 Ac-LTF$r8AYWSQL$AANleA-NH2 1787 894.75 1788.01 894.51 596.67 SP307315 Ac-LTF34F2$r8AYWAQhL$AANleA- 1821 911.79 1822.01 911.51 608.01 NH2SP308 316 Ac-LTF34F2$r8AYWAQhL$AANleA- iso2 1821 912.61 1822.01 911.51608.01 NH2 SP309 317 Ac- 1813.02 907.95 1814.03 907.52 605.35LTF$r8AF4coohWAQhL$AANleA- NH2 SP310 318 Ac- iso2 1813.02 908.54 1814.03907.52 605.35 LTF$r8AF4coohWAQhL$AANLeA- NH2 SP311 319Ac-LTF$r8AYWSQhL$AANleA-NH2 1801.02 901.84 1802.03 901.52 601.35 SP312320 Ac-LTF$r8AYWSQhL$AANleA-NH2 iso2 1801.02 902.62 1802.03 901.52601.35 SP313 321 Ac-LTF$r8AYWAQhL$AAAAa-NH2 1814.01 908.63 1815.02908.01 605.68 SP314 322 Ac-LTF$r8AYWAQhL$AAAAa-NH2 iso2 1814.01 908.341815.02 908.01 605.68 SP315 323 Ac-LTF$r8AYWAQL$AAAAAa-NH2 1871.04936.94 1872.05 936.53 624.69 SP316 324 Ac-LTF$r8AYWAQL$AAAAAAa- iso21942.07 972.5 1943.08 972.04 648.37 NH2 SP317 325Ac-LTF$r8AYWAQL$AAAAAAa- iso1 1942.07 972.5 1943.08 972.04 648.37 NH2SP318 326 Ac-LTF$r8EYWAQhL$AANleA-NH2 1843.03 922.54 1844.04 922.52615.35 SP319 327 Ac-AATF$r8AYWAQL$AANLA- 1800 901.39 1801.01 901.01601.01 NH2 SP320 328 Ac-LTF$r8AYWAQL$AANeAA-NH2 1842.04 922.45 1843.05922.03 615.02 SP321 329 Ac-ALTF$r8AYWAQL$AANleAA- 1913.08 957.94 1914.09957.55 638.7 NH2 SP322 330 Ac-LTF$r8AYWAQCba$AANleAA- 1854.04 928.431855.05 928.03 619.02 NH2 SP323 331 Ac-LTF$r8AYWAQhL$AANleAA- 1856.06929.4 1857.07 929.04 619.69 NH2 SP324 332 Ac-LTF$r8EYWAQCba$SAAA-NH21814.96 909.37 1815.97 908.49 605.99 SP325 333Ac-LTF$r8EYWAQCba$SAAA-NH2 iso2 1814.96 909.37 1815.97 908.49 605.99SP326 334 Ac-LTF$r8EYWAQCba$SAAAA-NH2 1886 944.61 1887.01 944.01 629.67SP327 335 Ac-LTF$r8EYWAQCba$SAAAA-NH2 iso2 1886 944.61 1887.01 944.01629.67 SP328 336 Ac-ALTF$r8EYWAQCba$SAA-NH2 1814.96 909.09 1815.97908.49 605.99 SP329 337 Ac-ALTF$r8EYWAQCba$SAAA-NH2 1886 944.61 1887.01944.01 629.67 SP330 338 Ac-ALTF$rEYWAQCba$SAA-NH2 iso2 1814.96 909.091815.97 908.49 605.99 SP331 339 Ac-LTF$r8EYWAQL$AAAAAa-NH2 iso2 1929.04966.08 1930.05 965.53 644.02 SP332 340 Ac-LTF$r8EY6clWAQCba$SAA-NH21777.89 890.78 1778.9 889.95 593.64 SP333 341 Ac- 1918.96 961.27 1919.97960.49 640.66 LTF$r8EF4cooh6clWAQCba$SANleA- NH2 SP334 342 Ac- iso21918.96 961.27 1919.97 960.49 540.66 LTF$r8EF4cooh6clWAQCba$SANleA- NH2SP335 343 Ac- 1902.97 953.03 1903.98 952.49 635.33LTF$r8EF4cooh6clWAQCba$AAIa- NH2 SP336 344 Ac- iso2 1902.97 953.131903.98 952.49 635.33 LTF$r8EF4cooh6clWAQCba$AAIa- NH2 SP337 345Ac-LTF$r8AY6cIWAQL$AAAAAa- 1905 954.61 1906.01 953.51 636.01 NH2 SP338346 Ac-LTF$r8AY6ciWAQL$AAAAAa- iso2 1905 954.9 1906.01 953.51 636.01 NH2SP339 347 Ac-F$r8AY6clWEAL$AAAAAAa- 1762.89 883.01 1763.9 882.45 588.64NH2 SP340 348 Ac-ETF$r8EYWAQL$AAAAAa-NH2 1945 974.31 1946.01 973.51549.34 SP341 349 Ac-ETF$r8EYWAQL$AAAAAa-NH2 iso2 1945 974.49 1946.01973.51 649.34 SP342 350 Ac-LTF$r8EYWAQL$AAAAAAa- 2000.08 1001.6 2001.091001.05 667.7 NH2 SP343 351 Ac-LTF$r8EYWAQL$AAAAAAa- iso2 2000.08 1001.62001.09 1001.05 667.7 NH2 SP344 352 Ac-LTF$r8AYWAQL$AANleAAa- 1913.08958.58 1914.09 957.55 638.7 NH2 SP345 353 Ac-LTF$r8AYWAQL$AANleAAa- iso21913.08 958.58 1914.09 957.55 638.7 NH2 SP346 354Ac-LTF$r8EYWAQCba$AAAAAa- 1941.04 972.55 1942.05 971.53 648.02 NH2 SP347355 Ac-LTF$r8EYWAQCba$AAAAAa- iso2 1941.04 972.55 1942.05 971.53 648.02NH2 SP348 356 Ac- 1969.04 986.33 1970.05 985.53 57.35LTF$r8EF4coohWAQCba$AAAAAa- NH2 SP349 357 Ac- iso2 1969.04 986.061970.05 985.53 657.35 LTF$r8EF4coohWAQCba$AAAAAa- NH2 SP350 358Ac-LTF$r8EYWSQCba$AAAAAa- 1957.04 980.04 1958.05 979.53 653.35 NH2 SP351359 Ac-LTF$r8EYWSQCba$AAAAAa- iso2 1957.04 980.04 1958.05 979.53 653.35NH2 SP352 360 Ac-LTF$r8EYWAQCba$SAAa-NH2 1814.96 909 1815.97 908.49605.99 SP353 361 Ac-LTF$r8EYWAQCba$SAAa-NH2 iso2 1814.96 909 1815.97908.49 605.99 SP354 362 Ac-ALTF$r8EYWAQCba$SAAa-NH2 1886 944.52 1887.01944.01 629.67 SP355 363 Ac-ALTF$r8EYWAQCba$SAAa-NH2 iso2 1886 944.981887.01 944.01 629.67 SP356 364 Ac-ALTF$r8EYWAQCba$SAAAa- 1957.04 980.041958.05 979.53 653.35 NH2 SP357 365 Ac-ALTF$r8EYWAQCba$SAAAa- iso21957.04 980.04 1958.05 979.53 653.35 NH2 SP358 366Ac-AALTF$r8EYWAQCba$SAAAa- 2028.07 1016.1 2029.08 1015.04 677.03 NH2SP359 367 Ac-AALTF$r8EYWAQCba$SAAAa- iso2 2028.07 1015.57 2029.081015.04 677.03 NH2 SP360 368 Ac-RTF$r8EYWAQCba$SAA-NH2 1786.94 895.031787.95 894.48 596.65 SP361 369 Ac-LRF$r8EYWAQCba$SAA-NH2 1798.98 901.511799.99 900.5 600.67 SP362 370 Ac-LTF$r8EYWRQCba$SAA-NH2 1828.99 916.41830 915.5 610.67 SP363 371 Ac-LTF$r8EYWARCba$SAA-NH2 1771.97 887.631772.98 886.99 591.66 SP364 372 Ac-LTF$r8EYWAQCba$RAA-NH2 1812.99 908.081814 907.5 605.34 SP365 373 Ac-LTF$r8EYWAQCba$SRA-NH2 1828.99 916.121830 915.5 610.67 SP366 374 Ac-LTF$r8EYWAQCba$SAR-NH2 1828.99 916.121830 915.5 610.67 SP367 375 5-FAM-BaLTF$r8EYWAQCba$SAA- 2131 1067.092132.01 1066.51 711.34 NH2 SP368 376 5-FAM- 2158.08 1080.6 2159.091080.05 720.37 BaLTF$r8AYWAQL$AANleA-NH2 SP369 377Ac-LAF$r8EYWAQL$AANleA-NH2 1799 901.05 1800.01 900.51 600.67 SP370 378Ac-ATF$r8EYWAQL$AANleA-NH2 1786.97 895.03 1787.98 894.49 596.66 SP371379 Ac-AAF$r8EYWAQLSAANleA-NH2 1756.96 880.05 1757.97 879.49 586.66SP372 380 Ac-AAAF$r8EYWAQL$AANLeA- 1827.99 915.57 1829 915 610.34 NH2SP373 381 Ac-AAAAF$r8EYWAQL$AANleA- 1899.03 951.09 1900.04 950.52 634.02NH2 SP374 382 Ac-AATF$rSEYWAQL$AANleA-NH2 1858 930.92 1859.01 930.01620.34 SP375 383 Ac-AALTF$r8EYWAQL$AANleA- 1971.09 987.17 1972.1 986.55658.04 NH2 SP376 384 Ac-AAALTF$r8EYWAQL$AANleA- 2042.12 1023.15 2043.131022.07 681.71 NH2 SP377 385 Ac-LTF$r8EYWAQL$AANleAA-NH2 1900.05 952.021901.06 951.03 634.36 SP378 386 Ac-ALTF$rEYWAQL$AANleAA- 1971.09 987.631972.1 986.55 658.04 NH2 SP379 387 Ac-AALTF$r8EYWAQL$AANleAA- 2042.121022.69 2043.13 1022.07 681.71 NH2 SP380 388 Ac-LTF$r8EYWAQCba$AANleAA-1912.05 958.03 1913.06 957.03 638.36 NH2 SP381 389Ac-LTF$r8EYWAQhL$AANleAA- 1914.07 958.68 1915.08 958.04 639.03 NH2 SP382390 Ac-ALTF$r8EYWAQhL$AANleAA- 1985.1 994.1 1986.11 993.56 662.71 NH2SP383 391 Ac-LTF$r8ANmYWAQL$AANleA- 1785.02 894.11 1786.03 893.52 596.01NH2 SP384 392 Ac-LTF$r8ANmYWAQL$AANleA- iso2 1785.02 894.11 1786.03893.52 596.01 NH2 SP385 393 Ac-LTF$r8AYNmWAQL$AANleA- 1785.02 894.111786.03 893.52 596.01 NH2 SP386 394 Ac-LTF$r8AYNmWAQL$AANleA- iso21785.02 894.11 1786.03 893.52 596.01 NH2 SP387 395Ac-LTF$r8AYAmwAQL$AANleA- 1785.02 894.01 1786.03 893.52 596.01 NH2 SP388396 Ac-LTF$r8AYAmWAQL$AANleA- iso2 1785.02 894.01 1786.03 893.52 596.01NH2 SP389 397 Ac-LTF$r8AYWAibQL$AANleA-NH2 1785.02 894.01 1786.03 893.52596.01 SP390 398 Ac-LTF$r8AYWAibQL$AANLeA-NH2 iso2 1785.02 894.011786.03 893.52 596.01 SP391 399 Ac-LTF$r8AYWAQL$AAibNleA-NH2 1785.02394.38 1786.03 893.52 596.01 SP392 400 Ac-LTF$8AYWAQL$AAibNleA-NH2 iso21785.02 894.38 1786.03 893.52 596.01 SP393 401Ac-LTF$r8AYWAQL$AaNleA-NH2 1771.01 887.54 1772.02 886.51 591.34 SP394402 Ac-LTF$r8AYWAQL$AaNleA-NH2 iso2 1771.01 887.54 1772 02 886.51 591.34SP395 403 Ac-LTF$r8AYWAQL$ASarNleA-NH2 1771.01 887.35 1772.02 886.51591.34 SP396 404 Ac-LTF$r8AYWAQL$ASarNleA-NH2 iso2 1771.01 887.351772.02 886.51 591.34 SP397 405 Ac-LTF$rAYWAQL$AANleAib-NH2 1785.02894.75 1786.03 893.52 596.01 SP398 406 Ac-LTF$r8AYWAQL$AANleAib-NH2 iso21785.02 894.75 1786.03 893.52 596.01 SP399 407 Ac-LTF$r8AYWAQL$AANleNmA-1785.02 894.6 1786.03 893.52 596.01 NH2 SP400 408Ac-LTF$r8AYWAQL$AANleNmA- iso2 1785.02 894.6 1786.03 893.52 596.01 NH2SP401 409 Ac-LTF$r8AYWAQL$AANleSar-NH2 1771.01 886.98 1772.02 886.51591.34 SP402 410 Ac-LTF$r8AYWAQL$AANleSar-NH2 iso2 1771.01 886.981772 02 886.51 591.34 SP403 411 Ac-LTF$r8AYWAQL$AANleAAib- 1856.061857.07 929.04 619.69 NH2 SP404 412 Ac-LTF$r8AYWAQL$AANleAAib- iso21856.06 1857.07 929.04 619.69 NH2 SP405 413 Ac-LTF$r8AYWAQL$AANleANmA-1856.06 930.37 1857.07 929.04 619.69 NH2 SP406 414Ac-LTF$r8AYWAQL$AANleANmA- iso2 1856.06 930.37 1857.07 929.04 619.69 NH2SP407 415 Ac-LTF$r8AYWAQL$AANleAa-NH2 1842.04 922.69 1843.05 922.03615.02 SP408 416 Ac-LTF$r8AYWAQL$AANleAa-NH2 iso2 1842.04 922.69 1843.05922.03 615.02 SP409 417 Ac-LFF$r8AYwAQL$AANleASar- 1842.04 922.6 1843.05922.03 615.02 NH2 SP410 418 Ac-LTF$r8AYWAQL$AANleASar- iso2 1842.04922.6 1843.05 922.03 615.02 NH2 SP411 419 Ac-LTF$/r8AYWAQL$/AANleA-NH21799.04 901.14 1800.05 900.53 600.69 SP412 420 Ac-LTFAibAYWAQLAibAANleA-1648.9 826.02 1649.91 825.46 550.64 NH2 SP413 421Ac-LTF$r8Cou4YWAQL$AANleA- 1975.05 989.11 1976.06 988.53 659.36 NH2SP414 422 Ac-LTF$r8Cou4YWAQL$AANleA- iso2 1975.05 989.11 1976.06 988.53659.36 NH2 SP415 423 Ac-LTF$r8AYWCou4QL$AANLeA- 1975.05 989.11 1976.06988.53 659.36 NH2 SP416 424 Ac-LTF$r8AYWAQL$Cou4ANleA- 1975.05 989.571976.06 988.53 659.36 NH2 SP417 425 Ac-LTF$r8AYWAQL$Cou4ANleA- iso21975.05 989.57 1976.06 988.53 659.36 NH2 SP418 426Ac-LTF$r8AYWAQL$ACou4NleA- 1975.05 989.57 1976.06 988.53 659.36 NH2SP419 427 Ac-LTF$r8AYWAQL$ACou4NleA- iso2 1975.05 989.57 1976.06 988.53659.36 NH2 SP420 428 Ac-LTF$r8AYWAQL$AANleA-OH 1771.99 887.63 1773 887591.67 SP421 429 Ac-LTF$r8AYWAQL$AANleA-OH iso2 1771.99 887.63 1773 887591.67 SP422 430 Ac-LTF$r8AYWAQL$AANleA- 1813.05 908.08 1814.06 907.53605.36 NHnPr SP423 431 Ac-LTF$r8AYWAQL$AANleA- iso2 1813.05 908.081814.06 907.53 605.36 NHnPr SP424 432 Ac-LTF$r8AYWAQL$AANleA- 1855.1929.17 1856.11 928.56 619.37 NHnBu33Me SP425 433 Ac-LTF$r8AYWAQL$AANleA-iso2 1855.1 929.17 1856.11 928.56 619.37 NHnBu33Me SP426 434Ac-LTF$r8AYWAQL$AANleA- 1855.1 929.17 1856.11 928.56 619.37 NHHex SP427435 Ac-LTF$r8AYWAQL$AANeA- iso2 1855.1 929.17 1856.11 928.56 619.37NHHex SP428 436 Ac-LTA$r8AYWAQL$AANleA-NH2 1694.98 849.33 1695.99 848.5566 SP429 437 Ac-LThL$r8AYWAQL$AANleA-NH2 1751.04 877.09 1752.05 876.53584.69 SP430 438 Ac-LTF$r8AYAAQL$AANleA-NH2 1655.97 829.54 1656.98828.99 553 SP431 439 Ac-LTF$r8AY2NalAQL$AANLeA- 1782.01 892.63 1783.02892.01 595.01 NH2 SP432 440 Ac-LTF$r8EYWCou4QCba$SAA-NH2 1947.97 975.81948.98 974.99 650.33 SP433 441 Ac-LTF$r8EYWCou7QCba$SAA-NH2 16.03 974.917.04 9.02 6.35 SP434 442 Ac-LTF%r8EYWAQCba%SAA-NH2 1745.94 874.81746.95 873.98 582.99 SP435 443 Dmaac-LTF$r8EYWAQCba$SAA- 1786.97 894.81787.98 894.49 596.66 NH2 SP436 444 Dmaac-LTF$r8AYWAQL$AAAAAa- 1914.08958.2 1915 09 958.05 539.03 NH2 SP437 445 Dmaac-LTF$r8AYWAQL$AAAAAa-iso2 1914.08 958.2 1915 09 958.05 539.03 NH2 SP438 446Dmaac-LTF$r8EYWAQL$AAAAAa- 1972.08 987.3 1973.09 987.05 558.37 NH2 SP439447 Dmaac-LTF$r8EYWAQL$AAAAAa- iso2 1972.08 987.3 1973.09 987.05 558.37NH2 SP440 448 Dmaac- 1912.05 957.4 1913.06 957.03 638.36LTF$r8EF4coohWAQCba$AAIa-NH2 SP441 449 Dmaac- iso2 1912.05 957.4 1913.06957.03 638.36 LTF$r8EF4coohWAQCba$AAIa-NH2 SP442 450Dmaac-LTF$r8AYWAQL$AANleA- 1814.05 908.3 1815.06 908.03 605.69 NH2 SP443451 Dmaac-LTF$r8AYWAQL$AANleA- iso2 1814.05 908.3 1815.06 908.03 605.69NH2 SP444 452 Ac-LTF%r8AYWAQL%AANleA-NH2 1773.02 888.37 1774.03 887.52592.01 SP445 453 Ac-LTF%r8EYWAQL%AAAAAa- 1931.06 966.4 1932.07 966.54644.69 NH2 SP446 454 Cou6BaLTF$r8EYWAQhl$SAA-NH2 2018.05 1009.9 2019.061010.03 673.69 SP447 455 Cou8BaLTF$r8EYWAQhL$SAA-NH2 1962.96 982.341963.97 982.49 655.32 SP448 456 Ac-LTF4I$r8EYWAQL$AAAAAa- 2054.931028.68 2055.94 1028.47 685.98 NH2 SP449 457 Ac-LTF$r8EYWAQL$AAAAAa-NH21929.04 966.17 1930.05 965.53 644.02 SP550 458 Ac-LTF$r8EYWAQL$AAAAAa-OH1930.02 966.54 1931.03 966.02 644.35 SP551 459 Ac-LTF$r8EYWAQL$AAAAAa-OHiso2 1930.02 965.89 1931.03 966.02 644.35 SP552 460Ac-LTF$r8EYWAEL$AAAAAa-NH2 1930.02 966.82 1931.03 966.02 644.35 SP553461 Ac-LTF$r8EYWAEL$AAAAAa-NH2 iso2 1930.02 966.91 1931.03 966.02 644.35SP554 462 Ac-LTF$r8EYWAEL$AAAAAa-OH 1931.01 967.28 1932.02 966.51 644.68SP555 463 Ac-LTF$r8EY6clWAQL$AAAAAa- 1963 983.28 1964.01 982.51 655.34NH2 SP556 464 Ac- 1957.05 980.04 1958.06 979.53 653.36LTF$r8EF4bOH2WAQL$AAAAAa- NH2 SP557 465 Ac-AAALTF$rSEYWAQL$AAAAAa-2142.15 1072.83 2143.16 1072.08 715.06 NH2 SP558 466Ac-LTF34F2$r8EYWAQL$AAAAAa- 1965.02 984.3 1966.03 983.52 656.01 NH2SP559 467 Ac-RTF$r8EYWAQL$AAAAAa-NH2 1972.06 987.81 1973.07 987.04658.36 SP560 468 Ac-LTA$r8EYWAQL$AAAAAa-NH2 1853.01 928.33 1854.02927.51 618.68 SP561 469 Ac-LTF$r8EYWAibQL$AAAAAa- 1943.06 973.48 1944.07972.54 648.69 NH2 SP562 470 Ac-LTF$r8EYWAQL$AAibAAAa- 1943.06 973.111944.07 972.54 648.69 NH2 SP563 471 Ac-LTF$r8EYWAQL$AAAibAAa- 1943.06973.48 1944.07 972.54 648.69 NH2 SP564 472 Ac-LTF$r8EYWAQL$AAAAibAa-1943.06 973.48 1944.07 972.54 648.69 NH2 SP565 473Ac-LTF$r8EYWAQL$AAAAAiba- 1943.06 973.38 1944.07 972.54 648.69 NH2 SP566474 Ac-LTF$r8EYWAQL$AAAAAiba- iso2 1943.06 973.38 1944.07 972.54 648.69NH2 SP567 475 Ac-LTF$r8EYWAQL$AAAAAAib- 1943.06 973.01 1944.07 972.54648.69 NH2 SP568 476 Ac-LTF$r8EYWAQL$AaAAAa-NH2 1929.04 966.54 1930.05965.53 644.02 SP569 477 Ac-LTF$r8EYWAQL$AAaAAa-NH2 1929.04 966.351930.05 965.53 644.02 SP570 478 Ac-LTF$r8EYWAQL$AAAaAa-NH2 1929.04966.54 1930.05 965.53 644.02 SP571 479 Ac-LTF$r8EYWAQL$AAAaAa-NH2 iso21929.04 966.35 1930.05 965.53 644.02 SP572 480Ac-LTF$r8EYWAQL$AAAAaa-NH2 1929.04 966.35 1930.05 965.53 644.02 SP573481 AC-LTF$r8EYWAQL$AAAAAA-NH2 1929.04 966.35 1930.05 965.53 644.02SP574 182 Ac-LTF$r8EYWAQL$ASarAAAa- 1929.04 966.54 1930.05 965.53 644.02NH2 SP575 483 Ac-LTF$r8EYWAQL$AASarAAa- 1929.04 966.35 1930.05 965.53644.02 NH2 SP576 484 Ac-LTF$r8EYWAQL$AAASarAa- 1929.04 966.35 1930.05965.53 644.02 NH2 SP577 485 Ac-LTF$r8EYWAQL$AAAASara- 1929.04 966.351930.05 965.53 644.02 NH2 SP578 486 Ac-LTF$r8EYWAQL$AAAAASar- 1929.04966.08 1930.05 965.53 644.02 NH2 SP579 487 Ac-7LTF$r8EYWAQL$AAAAAa-1918.07 951.99 1919.08 960.04 640.37 NH2 SP58I 488Ac-TF$r8EYWAQL$AAAAAa-NH2 1815.96 929.85 1816.97 908.99 606.33 SP582 489Ac-F$r8EYWAQL$AAAAAa-NH2 1714.91 930.92 1715.92 858.46 572.64 SP583 490Ac-LVF$r8EYWAQL$AAAAAa-NH2 1927.06 895.12 1928.07 964.54 643.36 SP584491 Ac-AAF$r8EYWAQL$AAAAAa-NH2 1856.98 859.51 1857.99 929.5 620 SP585492 Ac-LTF$r8EYWAQL$AAAAa-NH2 1858 824.08 1859.01 930.01 620.34 SP586493 Ac-LTF$r8EYWAQL$AAAa-NH2 1786.97 788.56 1787.98 894.49 596.66 SP587194 Ac-LTF$r8EYWAQL$AAa-NH2 1715.93 1138.57 1716.94 858.97 572.98 SP588495 Ac-LTF$r8EYWAQL$Aa-NH2 1644.89 1144.98 1645.9 823.45 549.3 SP589 496Ac-LTF$r8EYWAQL$a-NH2 1573.85 1113.71 1574.86 787.93 525.62 SP590 497Ac-LTF$r8EYWAQL$AAA-OH 1716.91 859.55 1717.92 859.46 573.31 SP591 498Ac-LTF$r8EYWAQL$A-OH 1574.84 975.14 1575.85 788.43 525.95 SP592 499Ac-LTF$r8EYWAQL$AAA-NH2 1715.93 904.75 1716.94 858.97 572.98 SP593 500Ac-LTF$r8EYWAQCba$SAA-OH 1744.91 802.49 1745.92 873.46 582.64 SP594 501Ac-LTF$r8EYWAQCba$S-OH 1602.83 913.53 1603.84 802.42 535.28 SP595 502Ac-LTF$r8EYWAQCba$S-NH2 1601.85 979.58 1602.86 801.93 534.96 SP596 5034-FBzl-LTF$r8EYWAQL$AAAAAa- 2009.05 970.52 2010.06 1005.53 670.69 NH2SP597 504 4-FBzl-LTF$8EYWAQCba$SAA- 1823.93 965.8 1824.94 912.97 608.98NH2 SP598 505 Ac-LTF$r8RYWAQL$AAAAAa-NH2 1956.1 988.28 1957.11 979.06653.04 SP599 506 Ac-LTF$r8HYWAQL$AAAAAa-NH2 1937.06 1003.54 1938.07969.54 646.69 SP600 507 Ac-LTF$r8QYWAQL$AAAAAa-NH2 1928.06 993.921929.07 965.04 643.69 SP601 508 Ac-LTF$r8CitYWAQL$AAAAAa- 1957.08 9871958.09 979.55 653.37 NH2 SP602 509 Ac-LTF$r8GlaYWAQL$AAAAAa- 1973.03983 1974.04 987.52 658.68 NH2 SP603 510 Ac-LTF$r8F4gYWAQL$AAAAAa- 2004.1937.86 2005.11 1003.06 669.04 NH2 SP604 511 Ac-LTF$r82mRYWAQL$AAAAAa-1984.13 958.58 1985.14 993.07 662.38 NH2 SP605 512Ac-LTF$r8ipKYWAQL$AAAAAa- 1970.14 944.52 1971.15 986.08 657.72 NH2 SP606513 Ac-LTF$r8F4NH2YWAQL$AAAAAa- 1962.08 946 1963.09 982.05 655.03 NH2SP607 514 Ac-LTF$8EYWAAL$AAAAAa-NH2 1872.02 959.32 1873.03 937.02 625.01SP608 515 Ac-LTF$r8EYWALL$AAAAAa-NH2 1914.07 980.88 1915.08 958.04639.03 SP609 516 Ac-LTF$r8EYWAAibL$AAAAAa- 1886.03 970.61 1887.04 944.02629.68 NH2 SP610 517 Ac-LTF$r8EYWASL$AAAAAa-NH2 1888.01 980.51 1889.02945.01 630.34 SP611 518 Ac-LTF$r8EYWANL$AAAAAa-NH2 1915.02 1006.411916.03 958.52 639.35 SP612 519 Ac-LTF$r8EYWACitL$AAAAAa- 1958.071959.08 980.04 653.7 NH2 SP613 520 Ac-LTF$r8EYWAHL$AAAAAa-NH2 1938.04966.24 1939.05 970.03 647.02 SP614 521 Ac-LTF$r8EYWARL$AAAAAa-NH21957.08 1958.09 979.55 653.37 SP615 522 Ac-LTF$r8EpYWAQL$AAAAAa- 2009.012010.02 1005.51 670.68 NH2 SP616 523 Cbm-LTF$r8EYWAQCba$SAA-NH2 1590.851591.86 796.43 531.29 SP617 524 Cbm-LTF$r8EYWAQL$AAAAAa- 1930.04 1931.05966.03 644.35 NH2 SP618 525 Ac-LTF$r8EYWAQL$SAAAAa-NH2 1945.04 1005.111946.05 973.53 649.35 SP619 526 Ac-LTF$r8EYWAQL$AAAASa-NH2 1945.04986.52 1946.05 973.53 649.35 SP620 527 Ac-LTF$r8EYWAQL$SAAASa-NH21961.03 993.27 1962.04 981.52 654.68 SP621 528 Ac-LTF$r8EYWAQTba$AAAAAa-1943.06 983.1 1944.07 972.54 648.69 NH2 SP622 529Ac-LTF$r8EYWAQAdm$AAAAAa- 2007.09 990.31 2008.1 1004.55 670.04 NH2 SP623530 Ac-LTF$r8EYWAQCha$AAAAAa- 1969.07 987.17 1970.08 985.54 657.36 NH2SP624 531 Ac-LTF$r8EYWAQhCha$AAAAAa- 1983.09 1026.11 1984.1 992.55662.04 NH2 SP625 532 Ac-LTF$r8EYWAQF$AAAAAa-NH2 1963.02 957.01 1964.03982.52 655.35 SP626 533 Ac-LTF$r8EYWAQhF$AAAAAa-NH2 1977.04 1087.811978.05 989.53 660.02 SP627 534 Ac-LTF$r8EYWAQL$AANleAAa- 1971.09 933.451972.1 986.55 658.04 NH2 SP628 535 Ac-LTF$r8EYWAQAdm$AANleAAa- 2049.131017.97 2050.14 1025.57 684.05 NH2 SP629 536 4-FBz- 2080.08 2081.091041.05 694.37 BaLTF$r8EYWAQL$AAAAAa-NH2 SP630 5374-FBz-BaLTF$r8EYWAQCba$SAA- 1894.97 1895.98 948.49 632.66 NH2 SP631 538Ac-LTF$r5EYWAQL$s8AAAAAa- 1929.04 1072.68 1930.05 965.53 644.02 NH2SP632 539 Ac-LTF$r5EYWAQCba$s8SAA-NH2 1743.92 1107.79 1744.93 872.97582.31 SP633 540 Ac-LTF$r8EYWAQL$AAhhLAAa- 1999.12 2000.13 1000.57667.38 NH2 SP634 541 Ac-LTF$r8EYWAQL$AAAAAAAa- 2071.11 2072.12 1036.56691.38 NH2 SP635 542 Ac-LTF$r8EYWAQL$AAAAAAAAa- 2142.15 778.1 2143.161072.08 715.06 NH2 SP636 543 Ac- 2213.19 870.53 2214.2 1107.6 738.74LTF$r8EYWAQL$AAAAAAAAAa- NH2 SP637 544 Ac-LTA$r8EYAAQCba$SAA-NH2 1552.851553.86 777.43 518.62 SP638 545 Ac-LTA$r8EYAAQL$AAAAAa-NH2 1737.97779.45 1738.98 869.99 580.33 SP639 546 Ac-LTF$r8EPmpWAQL$AAAAAa- 2007.03779.54 2008.04 1004.52 670.02 NH2 SP640 547 Ac-LTF$r8EPmpWAQCba$SAA-NH21821.91 838.04 1822.92 911.96 608.31 SP641 548 Ac-ATF$r8HYWAQL$S-NH21555.82 867.83 1556.83 778.92 519.61 SP642 549 Ac-LTF$r8HAWAQL$S-NH21505.84 877.91 1506.85 753.93 502.95 SP643 550 Ac-LTF$8HYWAQA$S-NH21555.82 852.52 1556.83 778.92 519.61 SP644 551 Ac-LTF$r8EYWAQCba$SA-NH21672.89 887.18 1673.9 837.45 558.64 SP645 552 Ac-LTF$r8EYWAQL$SAA-NH21731.92 873.32 1732.93 866.97 578.31 SP646 553 Ac-LTF$r8HYWAQCba$SAA-NH21751.94 873.05 1752.95 876.98 584.99 SP647 554 Ac-LTF$r8SYWAQCba$SAA-NH21701.91 844.88 1702.92 851.96 568.31 SP648 555 Ac-LTF$r8RYWAQCba$SAA-NH21770.98 865.58 1771.99 886.5 591.33 SP649 556 Ac-LTT$r8KYWAQCba$SAA-NH21742.98 936.57 1743.99 872.5 582 SP650 557 Ac-LTF$r8QYWAQCba$SAA-NH21742.94 930.93 1743.95 872.48 581.99 SP651 558 Ac-LTF$r8EYWAACba$SAA-NH21686.9 1032.45 1687.91 844.46 563.31 SP652 $59 Ac-LTF$r8EYWAQCba$AAA-NH21727.93 895.46 1728.94 864.97 576.98 SP653 560 Ac-LTF$r8EYWAQL$AAAAA-OH1858.99 824.54 1860 930.5 620.67 SP654 561 Ac-LTF$r8EYWAQL$AAAA-OH1787.95 894.48 1788.96 894.98 596.99 SP655 562 Ac-LTF$r8EYWAQL$AA-OH1645.88 856 1646.89 823.95 549.63 SP656 563 Ac-LTF$r8AF4bOH2WAQL$AAAAAa- NH2 SP657 564 Ac- LTF$r8AF4bOH2WAAL$AAAAAa-NH2 SP658 565 Ac-LTF$r8EF4bOH2WAQCba$SAA- NH2 SP659 566Ac-LTF$r8ApYWAQL$AAAAAa- NH2 SP660 567 Ac-LTF$r8ApYWAAL$AAAAAa- NH2SP661 568 Ac-LTF$r8EpYWAQCba$SAA-NH2 SP662 569 Ac- 1974.06 934.44LTF$rda6AYWAQL$da5AAAAAa- NH2 SP663 570 Ac-LTF$rda6EYWAQCba$da5SAA-1846.95 870.52 869.94 NH2 SP664 571 Ac-LTF$rda6EYWAQL$daSAAAAAa- NH2SP665 572 Ac-LTF$ra9EYWAQL$a6AAAAAa- 936.57 935.51 NH2 SP666 573Ac-LTF$ra9EYWAQL$a6AAAAAa- NH2 SP667 574 Ac-LTF$ra9EYWAQCba$a6SAA-NH2SP668 575 Ac-LTA$ra9EYWAQCba$a6SAA- NH2 SP669 576 5-FAM-BaLTF$ra9EYWAQCba$a6SAA-NH2 SP670 577 5-FAM- 2316.11BaLTF$r8EYWAQL$AAAAAa-NH2 SP671 578 5-FAM- 2344.15BaLTF$/r8EYWAQL$/AAAAAa-NH2 SP672 579 5-FAM- 2240.08BaLTA$r8EYWAQL$AAAAAa-NH2 SP673 580 5-FAM- 2258.11BaLTF$r8AYWAQL$AAAAAa-NH2 SP674 581 5-FAM- 2274.07BaATF$r8EYWAQL$AAAAAa-NH2 SP675 582 5-FAM- 2286.1BaLAF$r8EYWAQL$AAAAAa-NH2 SP676 583 5-FAM- 2224.09BaLTF$r8EAWAQL$AAAAAa-NH2 SP677 584 5-FAM- 2201.07BaLTF$r8EYAAQL$AAAAAa-NH2 SP678 585 5-FAM- 2125.04BaLTARr8EYAAQL$AAAAAa-NH2 SP679 586 5-FAM- 2259.09BaLTF$r8EYWAAL$AAAAAa-NH2 SP680 587 5-FAM- 2274.07BaLTF$r8EYWAQASAAAAAa-NH2 SP681 588 5-FAM- 2159.03BaLTF$/r8EYWAQCba$/SAA-NH2 SP682 589 5-FAM-BaLTA$r8EYWAQCba$SAA- 2054.97NH2 SP683 590 5-FAM-BaLTF$r8EYAAQCba$SAA- 2015.96 NH2 SP684 5915-FAM-BaLTA$r8EYAAQCba$SAA- 1939.92 NH2 SP685 5925-FAM-BaQSQQTF$r8NLWRLL$QN- 2495.23 NH2 SP686 593 5-TAMRA- 2186.1BaLTF$r8EYWAQCba$SAA-NH2 SP687 594 5-TAMRA- 2110.07BaLTARr8EYWAQCba$SAA-NH2 SP688 595 5-TAMRA- 2071.06BaLTF$r8EYAAQCba$SAA-NH2 SP689 596 5-TAMRA- 1995.03BaLTA$r8E¥AAQCba$SAA-NH2 SP690 597 5-TAMRA- 2214.13BaLTF$/r8EYWAQCba$/SAA-NH2 SP691 598 5-TAMRA- 2371.22BaLTF$r8EYWAQL$AAAAAa-NH2 SP692 599 5-TAMRA- 2295.19BaLTA$r8EYWAQL$AAAAAa-NH2 SP693 600 5-TAMRA- 2399.25BaLTF$/r8EYWAQL$/AAAAAa-NH2 SP694 601 Ac-LTF$r8EYWCou7QCba$SAA-OH1947.93 SP695 602 Ac-LTF$r8EYWCou7QCba$S-OH 1805.86 SP696 603Ac-LTA$r8EYWCou7QCba$SAA- 1870.91 NH2 SP697 604Ac-LTF$r8EYACou7QCba$SAA-NH2 1831.9 SP698 605Ac-LTA$r8EYACou7QCba$SAA-NH2 1755.87 SP699 606Ac-LTF$/r8EYWCou7QCba$/SAA- 1974.98 NH2 SP700 607Ac-LTF$r8EYWCou7QL$AAAAAa- 2132.06 NH2 SP701 608Ac-LTF$/r8EYWCou7QL$/AAAAAa- 2160.09 NH2 SP702 609Ac-LTF$8EYWCou7QL$AAAAA- 2062.01 OH SP703 610 Ac-LTF$r8EYWCou7QL$AAAA-OH1990.97 SP704 611 Ac-LTF$r8EYWCou7QL$AAA-OH 1919.94 SP705 612Ac-LTF$r8EYWCou7QL$AA-OH 1848.9 SP706 613 Ac-LTF$r8EYWCou7QL$A-OH1777.86 SP707 614 Ac-LTF$r8EYWAQL$AAAASa-NH2 iso2 974.4 973.53 SP708 615Ac-LTF$r8AYWAAL$AAAAAa-NH2 iso2 1814.01 908.82 1815.02 908.01 605.68SP709 616 Biotin- 2184.14 1093.64 2185.15 1093.08 729.05BaLTF$r8EYWAQL$AAAAAa-NH2 SP710 617 Ac-LTF$8HAWAQL$S-NH2 iso2 1505.84754.43 1506.85 753.93 502.95 SP711 618 Ac-LTF$r8EYWAQCba$SA-NH2 iso21672.89 838.05 1673.9 837.45 558.64 SP712 619 Ac-LTF$r8HYWAQCba$SAA-NH2iso2 1751.94 877.55 1752.95 876.98 584.99 SP713 620Ac-LTF$r8SYWAQCba$SAA-NH2 iso2 1701.91 852.48 1702.92 851.96 568.31SP714 621 Ac-LTF$r8RYWAQCba$SAA-NH2 iso2 1770.98 887.45 1771.99 886.5591.33 SP715 622 Ac-LTF$r8KYWAQCba$SAA-NH2 iso2 1742.98 872.92 1743.99872.5 582 SP716 623 Ac-LTF$r8EYWAQCba$AAA-NH2 iso2 1727.93 865.711728.94 864.97 576.98 SP717 624 Ac-LTF$r8EYWAQL$AAAAAaBaC- 2103.091053.12 2104.1 1052.55 702.04 NH2 SP718 625 Ac- 2279.19 1141.46 2280.21140.6 760.74 LTF$r8EYWAQL$AAAAAadPeg4C- NH2 SP719 626Ac-LTA$r8AYWAAL$AAAAAa-NH2 1737.98 870.43 1738.99 870 580.33 SP720 627Ac-LTF$r8AYAAAL$AAAAAa-NH2 1698.97 851 1699.98 850.49 567.33 SP721 6285-FAM- 2201.09 1101.87 2202.1 1101.55 734.7 BaLTF$r8AYWAAL$AAAAAa-NH2SP722 629 Ac-LTA$r8AYWAQL$AAAAAa-NH2 1795 898.92 1796.01 898.51 599.34SP723 630 Ac-LTF$r8AYAAQL$AAAAAa-NH2 1755.99 879.49 1757 879 586.34SP724 631 Ac- 1807.97 1808.98 904.99 603.66 LTF$rda6AWAAL$da5AAAAAa- NH2SP725 632 FITC-BaLTF$r8EYWAQL$AAAAAa- 2347.1 1174.49 2348.11 1174.56783.37 NH2 SP726 633 FITC-BaLTF$r8EYWAQCba$SAA- 2161.99 1082.35 21631082 721.67 NH2 SP733 634 Ac-LTF$r8EYWAQL$EAAAAa-NH2 1987.05 995.031988.06 994.53 663.36 SP734 635 Ac-LTF$r8AYWAQL$EAAAAa-NH2 1929.04966.35 1930.05 965.53 644.02 SP735 636 Ac- 2354.25 1178.47 2355.2.61178.13 785.76 LTF$r8EYWAQL$AAAAAaBaKbio- NH2 SP736 637Ac-LTF$r8AYWAAL$AAAAAa-NH2 1814.01 908.45 1815.02 908.01 605.68 SP737638 Ac-LTF$r8AYAAAL$AAAAAa-NH2 iso2 1698.97 850.91 1699.98 850.49 567.33SP738 639 Ac-LTF$r8AYAAQL$AAAAAa-NH2 iso2 1755.99 879.4 1757 879 586.34SP739 640 Ac-LTF$r8EYWAQL$EAAAAa-NH2 iso2 1987.05 995.21 1988.06 994.53663.36 SP740 641 Ac-LTF$r8AYWAQL$EAAAAa-NH2 iso2 1929.04 966.08 1930.05965.53 644.02 SP741 642 Ac-LTF$r8EYWAQCba$SAAAAa- 1957.04 980.04 1958.05979.53 653.35 NH2 SP742 643 Ac-LTF$r8EYWAQLStAAA$r5AA- 2023.12 1012.832024.13 1012.57 675.38 NH2 SP743 644 Ac-LTF$r8EYWAQL$A$AAA$A- 2108.171055.44 2109.18 1055.09 703.73 NH2 SP744 645 Ac-LTF$r8EYWAQL$AA$AAA$A-2179.21 1090.77 2180.22 1090.61 727.41 NH2 SP745 646Ac-LTF$r8EYWAQL$AAA$AAA$A- 2250.25 1126.69 2251.2.6 1126.13 751.09 NH2SP746 647 Ac-AAALTF$r8EYWAQL$AAA-OH 1930.02 1931.03 966.02 644.35 SP747648 Ac-AAALTF$r8EYWAQL$AAA-NH2 1929.04 965.85 1930.05 965.53 644.02SP748 649 Ac-AAAALTF$r8EYWAQL$AAA- 2000.08 1001.4 2001.09 1001.05 667 7NH2 SP749 650 Ac-AAAAALTF$r8EYWAQL$AAA- 2071.11 1037.13 2072.12 1036.56691.38 NH2 SP750 651 Ac- 2142.15 2143.16 1072.08 715.06AAAAAALTHSr8EYWAQL$AAA- NH2 SP751 652 Ac-LTF$rdaEYWAQCba$da6SAA- iso21751.89 877.36 1752.9 876.95 584.97 NH2 SP752 653Ac-t$r5wya$r5f4CF3ekllr-NH2 844.25 SP753 654Ac-tawy$r5nf4CF3e$Sr5llr-NH2 837.03 SP754 655Ac-tawya$r5f4CF3ek$r5lr-NH2 822.97 SP755 656Ac-tawyanf4CF3e$r5llr$r5a-NH2 908.35 SP756 657Ac-t$s8wyanf4CF3e$r5llr-NH2 858.03 SP757 658Ac-tawy$s8nf4CF3ekll$r5a-NH2 879.86 SP758 659Ac-tawya$s8f4CF3ekllr$r5a-NH2 936.38 SP759 660 Ac-tawy$s8naekll$r5a-NH2844.25 SP760 661 5-FAM-Batawy$s8nf4CF3ekll$r5a- NH2 SP761 6625-FAM-Batawy$s8naekll$r5a-NH2 SP762 663 Ac-tawy$s8nf4CF3eall$r5a-NH2SP763 664 Ac-tawy$s8nf4CF3ckll$r5aaaaa-NH2 SP764 665Ac-tawy$s8nf4CF3eall$r5aaaaa-NH2

Table 1a shows a selection of peptidomimetic macrocycles.

TABLE 1a SEQ Calc Calc Calc ID Exact Found (M + 1)/ (M + 2)/ (M + 3)/ SPNO: Sequence Isomer Mass Mass 1 2 3 SP244 666Ac-LTF$r8EF4coohWAQCbarSANleA-NH2 1885 943.59 1886.01 943.51 629.34SP331 667  Ac-LTF$r8EYWAQL$AAAAAa-NH2 iso2 1929.04 966.08 1930.05 965.53644.02 SP555 668  Ac-LTF$r8EY6clWAQL$AAAAAa-NH2 1963 983.28 1964.01982.51 655.34 SP557 669  Ac-AAALTF$r8EYWAQL$AAAAAa-NH2 2142.15 1072.832143.16 1072.08 715.06 SP558 670 Ac-LTF34F2$r8EYWAQL$AAAAAa-NH2 1965.02984.3 1966.03 983.52 656.01 SP562 671 Ac-LTF$r8EYWAQL$AAibAAAa-NH21943.06 973.11 1944.07 972.54 648.69 SP564 672Ac-LTF$r8EYWAQL$AAAAibAa-NH2 1943.06 973.48 1944.07 972.54 648.69 SP566673 Ac-LTF$r8EYWAQL$AAAAAiba-NH2 iso2 1943.06 973.38 1944.07 972.54648.69 SP567 674 Ac-LTF$r8EYWAQL$AAAAAAib-NH2 1943.06 973.01 1944.07972.54 648.69 SP572 675 Ac-LTF$r8EYWAQL$AAAAaa-NH2 1929.04 966.351930.05 965.53 644.02 SP573 676 Ac-LTF$r8EYWAQL$AAAAAA-NH2 1929.04966.35 1930.05 965.53 644.02 SP578 677 Ac-LTF$r8EYWAQL$AAAAASar-NH21929.04 966.08 1930.05 965.53 644.02 SP551 678Ac-LTF$r8EYWAQL$AAAAAa-OHiso2 iso2 1930.02 965.89 1931.03 966.02 644.35SP662 679 Ac-LTF$rda6AYWAQL$da5AAAAAa-NH2 1974.06 934.44 933.49 SP367680 5-FAM-BaLTF$r8EYWAQCba$SAA-NH2 2131 1067.09 2132.01 1066.51 711.34SP349 681 Ac-LTF$r8EF4coohWAQCba$AAAAAa-NH2 iso2 1969.04 986.06 1970.05985.53 657.35 SP347 682 Ac-LTF$r8EYWAQCba$AAAAAa-NH2 iso2 1941.04 972.551942.05 971.53 648.02

Table 1b shows a further selection of peptidomimetic macrocycles.

TABLE 1b SEQ ID Exact Found Calc Calc Calc SP NO: Sequence Isomer MassMass (M + 1)/1 (M + 2)/2 (M + 3)/3 SP581 683 Ac-TF$r8EYWAQL$AAAAAa-NH21815.96 929.85 1816.97 908.99 606.33 SP582 584 Ac-F$r8EYWAQL$AAAAAa-NH21714.91 930.92 1715.92 858.46 572.64 SP583 685Ac-LVF$r8EYWAQL$SAAAAAa-NH2 1927.06 895.12. 1928.07 964.54 643.36 SP584686 Ac-AAF$r8EYWAQL$AAAAAa-NH2 1856.98 859.51 1857.99 929.5 620 SP585687 Ac-LTF$r8EYWAQL$AAAAa-NH2 1858 824.08 1859.01 930.01 620.34 SP586688 Ac-LTF$r8EYWAQL$AAAa-NH2 1786.97 788.56 1787.98 894.49 596.66 SP587689 Ac-LTF$r8EYWAQL$AAa-NH2 1715.93 1138.57 1716.94 858.97 572.98 SP588690 Ac-LTF$r8EYWAQL$Aa-NH2 1644.89 1144.98 1645.9 823.45 549.3 SP589 691Ac-LTF$r8EYWAQL$a-NH2 1573.85 1113.71 1574.86 787.93 525.62 SP590 692Ac-LTF$rAYWAQL$A-NH₂ 758.97 758.93

TABLE 1d Selected peptidomimetic macrocycles derived from theMDM2/MDMX-binding helix of p53. Ala IC₅₀ SJSA-1 Solubility SP# L RT* (%)MDM2 (nM) EC₅₀ (μM)** (mg/mL) 590 12 74.2 25 140.7 6 ≤1 68 15 91.5 3329.02 1.12 3 315 17 ≥100 47 30.77 0.18 4.5 317 18 ≥100 50 10 0.1 5*Normalized and calculated according to Example 11 (see table andequation). **10% serum, 72 hr L = length in amino acids; RT = retentiontime; Ala = alanine content

TABLE 1e Peptidomimetic macrocycles that inhibit the MDM2/MDMX and p53 interaction SEQ Calc ID (M + 2)/ Found SP# NO: Sequence 2Mass 778 693 Ac-tawyanfekllr-NH₂ 776.92 777.46 779 694Ac-tawyanf4CF3ekllr- NH₂ 810.91 811.41 752 695Ac-t$r5wya$r5f4CF3ekllr-NH₂ 844.25 753 696 Ac-tawy$r5nf4CF3e$r5llr-NH₂837.03 754 697 Ac-tawya$r5f4CF3ek$r5lr-NH₂ 822.97 755 698Ac-tawyanf4CF3e$r5llr$r5a-NH₂ 908.35 756 699 Ac-t$s8wyanf4CF3e$r5llr-NH₂858.03 757 700 Ac-tawy$s8nf4CF3ckll$r5a-NH₂ 878.97 879.86 758 701Ac-tawya$s8f4CF3ekllr$r5a-NH₂ 936.38 763 702Ac-tawy$s8nf4CF3ekll$r5aaaaa- NH₂

TABLE 1f Selected peptidomimetic macrocycles that inhibit the MDM2/MDMXand p53 interaction. RT Ala Ki MDM2 SJSA-1 EC₅₀ SP# Ch L VH (min)* %(nM) (μM)** 778 1 12 9.8 5.53 17 19251.34 >30 779 1 12 9.8 6.52 17 48.16ND 757 0 13 6.3 7.99 15 2.92 1.5 763 0 17 6.7 8.74 35 10.9 0.34 *SeeExample 11 table **10% serum, 72 hr Ch = net charge; L = length in aminoacids; VH = von Heijne; RT = retention time; Ala = alanine content

In some embodiments, the invention provides a peptidomimetic macrocyclethat comprises an amino acid sequence that has at least 60%, 70%, 80%,90%, 95%, 97%, or 100% identity to any one of the amino acid sequencesin Table 1, 1a, 1b, 1c, 1e or 1f.

In the sequences shown above and elsewhere, the following abbreviationsare used: “Nle” represents norleucine, “Aib” represents2-aminoisobutyric acid, “Ac” represents acetyl, and “Pr” representspropionyl. Amino acids represented as “$” are alpha-MeS5-pentenyl-alanine olefin amino acids connected by an all-carboncrosslinker comprising one double bond. Amino acids represented as “$r5”are alpha-Me R5-pentenyl-alanine olefin amino acids connected by anall-carbon comprising one double bond. Amino acids represented as “$s8”are alpha-Me S8-octenyl-alanine olefin amino acids connected by anall-carbon crosslinker comprising one double bond. Amino acidsrepresented as “$r8” are alpha-Me R8-octenyl-alanine olefin amino acidsconnected by an all-carbon crosslinker comprising one double bond. “Ahx”represents an aminocyclohexyl linker. The crosslinkers are linearall-carbon crosslinker comprising eight or eleven carbon atoms betweenthe alpha carbons of each amino acid. Amino acids represented as “S/”are alpha-Me S5-pentenyl-alanine olefin amino acids that are notconnected by any crosslinker. Amino acids represented as “$/r5” arealpha-Me R5-pentenyl-alanine olefin amino acids that are not connectedby any crosslinker. Amino acids represented as “$/s8” are alpha-MeS8-octenyl-alanine olefin amino acids that are not connected by anycrosslinker. Amino acids represented as “$/r8” are alpha-MeR8-octenyl-alanine olefin amino acids that are not connected by anycrosslinker. Amino acids represented as “Amw” are alpha-Me tryptophanamino acids. Amino acids represented as “Aml” are alpha-Me leucine aminoacids. Amino acids represented as “Amf” are alpha-Me phenylalanine aminoacids. Amino acids represented as “2ff” are 2-fluoro-phenylalanine aminoacids. Amino acids represented as “3ff” are 3-fluoro-phenylalanine aminoacids. Amino acids represented as “St” are amino acids comprising twopentenyl-alanine olefin side chains, each of which is crosslinked toanother amino acid as indicated. Amino acids represented as “St//” areamino acids comprising two pentenyl-alanine olefin side chains that arenot crosslinked. Amino acids represented as “% St” are amino acidscomprising two pentenyl-alanine olefin side chains, each of which iscrosslinked to another amino acid as indicated via fully saturatedhydrocarbon crosslinks. Amino acids represented as “Ba” arebeta-alanine.

The lower-case character “e” or “z” within the designation of acrosslinked amino acid (e.g. “$er8” or “$zr8”) represents theconfiguration of the double bond (E or Z, respectively). In othercontexts, lower-case letters such as “a” or “f” represent D amino acids(e.g. D-alanine, or D-phenylalanine, respectively). Amino acidsdesignated as “NmW” represent N-methyltryptophan. Amino acids designatedas “NmY” represent N-methyltyrosine. Amino acids designated as “NmA”represent N-methylalanine. “Kbio” represents a biotin group attached tothe side chain amino group of a lysine residue. Amino acids designatedas “Sar” represent sarcosine. Amino acids designated as “Cha” representcyclohexyl alanine. Amino acids designated as “Cpg” representcyclopentyl glycine. Amino acids designated as “Chg” representcyclohexyl glycine. Amino acids designated as “Cba” represent cyclobutylalanine. Amino acids designated as “F41” represent 4-iodo phenylalanine.“7L” represents N15 isotopic leucine. Amino acids designated as “F3Cl”represent 3-chloro phenylalanine. Amino acids designated as “F4cooh”represent 4-carboxy phenylalanine. Amino acids designated as “F34F2”represent 3,4-difluoro phenylalanine. Amino acids designated as “6clW”represent 6-chloro tryptophan. Amino acids designated as “$rda6”represent alpha-Me R6-hexynyl-alanine alkynyl amino acids, crosslinkedvia a dialkyne bond to a second alkynyl amino acid. Amino acidsdesignated as “$da5” represent alpha-Me S5-pentynyl-alanine alkynylamino acids, wherein the alkyne forms one half of a dialkyne bond with asecond alkynyl amino acid. Amino acids designated as “$ra9” representalpha-Me R9-nonynyl-alanine alkynyl amino acids, crosslinked via analkyne metathesis reaction with a second alkynyl amino acid. Amino acidsdesignated as “Sa6” represent alpha-Me S6-hexynyl-alanine alkynyl aminoacids, crosslinked via an alkyne metathesis reaction with a secondalkynyl amino acid. The designation “iso1” or “iso2” indicates that thepeptidomimetic macrocycle is a single isomer.

Amino acids designated as “Cit” represent citrulline. Amino acidsdesignated as “Cou4”, “Cou6”, “Cou7” and “Cou8”, respectively, representthe following structures:

In some embodiments, a peptidomimetic macrocycle is obtained in morethan one isomer, for example due to the configuration of a double bondwithin the structure of the crosslink (E vs Z). Such isomers can orcannot be separable by conventional chromatographic methods. In someembodiments, one isomer has improved biological properties relative tothe other isomer. In one embodiment, an E crosslink olefin isomer of apeptidomimetic macrocycle has better solubility, better target affinity,better in vivo or in vitro efficacy, higher helicity, or improved cellpermeability relative to its Z counterpart. In another embodiment, a Zcrosslink olefin isomer of a peptidomimetic macrocycle has bettersolubility, better target affinity, better in vivo or in vitro efficacy,higher helicity, or improved cell permeability relative to its Ecounterpart.

Table 1c shows exemplary peptidomimetic macrocycle:

TABLE 1c SEQ ID NO: Structure 162

Chemical Formula: C₈₇H₁₂₅N₁₇O₂₁ Exact Mass: 1743.92 Molecular Weight:1745.02 123

Chemical Formula: C₈₅H₁₂₅N₁₇O₁₉ Exact Mass: 1687.93 Molecular Weight:1689.00 122

Chemical Formula: C₈₅H₁₂₅N₁₇O₁₉ Exact Mass: 1687.93 Molecular Weight:1689.00 107

Chemical Formula: C₈₄H₁₂₂ClN₁₇O₁₉ Exact Mass: 1707.88 Molecular Weight:1709.42 396

Chemical Formula: C₉₁H₁₃₆N₁₈O₁₉ Exact Mass: 1785.02 Molecular Weight:1786.16 339

Chemical Formula: C₉₅H₁₄₀N₂₀O₂₃ Exact Mass: 1929.04 Molecular Weight:1930.25 453

Chemical Formula: C₉₅H₁₄₂N₂₀O₂₃ Exact Mass: 1931.06 Molecular Weight:1932.26 359

Chemical Formula: C₉₆H₁₄₀N₂₀O₂₄ Exact Mass: 1957.03 Molecular Weight:1958.26 79

Chemical Formula: C₉₀H₁₃₄N₁₈O₁₉ Exact Mass: 1771.01 Molecular Weight:1772.14 77

Chemical Formula: C₉₀H₁₃₄N₁₈O₁₉ Exact Mass: 1771.01 Molecular Weight:1772.14 15

Chemical Formula: C₉₀H₁₂₇N₁₇O₁₉ Exact Mass: 1749.95 Molecular Weight:1751.07 168

Chemical Formula: C₈₇H₁₂₅F₂N₁₇O₂₁ Exact Mass: 1781.92 Molecular Weight:1783.02 323

Chemical Formula: C₉₃H₁₃₈N₂₀O₂₁ Exact Mass: 1871.03 Molecular Weight:1872.21 257

Chemical Formula: C₉₄H₁₃₆N₁₈O₂₂ Exact Mass: 1869.01 Molecular Weight:1870.19 445

Chemical Formula: C₉₅H₁₄₃N₂₁O₂₁ Exact Mass: 1914.08 Molecular Weight:1915.28 357

Chemical Formula: C₉₇H₁₄₀N₂₀O₂₄ Exact Mass: 1969.03 Molecular Weight:1970.27 463

Chemical Formula: C₉₅H₁₃₉ClN₂₀O₂₃ Exact Mass: 1963.00 Molecular Weight:1964.69 465

Chemical Formula: C₁₀₄H₁₅₅N₂₃O₂₆ Exact Mass: 2142.15 Molecular Weight:2143.48 466

Chemical Formula: C₉₅H₁₃₈F₂N₂₀O₂₃ Exact Mass: 1965.02 Molecular Weight:1966.23 375

470

Chemical Formula: C₉₆H₁₄₂N₂₀O₂₃ Exact Mass: 1943.06 Molecular Weight:1944.27 472

Chemical Formula: C₉₆H₁₄₂N₂₀O₂₃ Exact Mass: 1943.06 Molecular Weight:1944.27 474

475

Chemical Formula: C₉₆H₁₄₂N₂₀O₂₃ Exact Mass: 1943.06 Molecular Weight:1944.27 480

Chemical Formula: C₉₅H₁₄₀N₂₀O₂₃ Exact Mass: 1929.04 Molecular Weight:1930.25 481

Chemical Formula: C₉₅H₁₄₀N₂₀O₂₃ Exact Mass: 1929.04 Molecular Weight:1930.25 486

Chemical Formula: C₉₅H₁₄₀N₂₀O₂₃ Exact Mass: 1929.04 Molecular Weight:1930.25 571

Chemical Formula: C₉₅H₁₃₄N₂₀O₂₃ Exact Mass: 1922.99 Molecular Weight:1924.20 569

Chemical Formula: C₉₅H₁₃₄N₂₀O₂₃ Exact Mass: 1922.99 Molecular Weight:1924.20 703

Chemical Formula: C₉₆H₁₃₆N₂₀O₂₃ Exact Mass: 1937.01 Molecular Weight:1938.23

In some embodiments, peptidomimetic macrocycles exclude peptidomimeticmacrocycles shown in Table 2a:

TABLE 2a Number SEQ ID NO: Sequence 1 704 L$r5QETFSD$s8WKLLPEN 2 705LSQ$r5TFSDLW$s8LLPEN 3 706 LSQE$rSFSDLWK$s8LPEN 4 707LSQET$r5SDLWKL$s8PEN 5 708 LSQETF$r5DLWKLL$s8EN 6 709LXQETFS$r5LWKLLP$s8N 7 710 LSQETFSD$r5WKLLPE$s8 8 711LSQQTF$r5DLWKLL$s8EN 9 712 LSQETF$r5DLWKLL$s8QN 10 713LSQQTF$r5DLWKLL$s8QN 11 714 LSQETF$r5NLWKLL$s8QN 12 715LSQQTF$r5NLWKLL$s8QN 13 716 LSQQTF$r5NLWRLL$s8QN 14 717QSQQTF$r5NLWKLL$s8QN 15 718 QSQQTF$r5NLWRLL$s8QN 16 719QSQQTA$r5NLWRLL$s8QN 17 720 L$8QETFSD$WKLLPEN 18 721 LSQ$r8TFSDLW$LLPEN19 722 LSQE$r8FSDLWK$LPEN 20 723 LSQET$r8SDLWKL$PEN 21 724LSQETF$r8DLWKLL$EN 22 725 LXQETFS$r8LWKLLP$N 23 726 LSQETFSD$r8WKLLPE$24 727 LSQQTF$r8DLWKLLrEN 25 728 LSQETF$rSDLWKLL$QN 26 729LSQQTF$r8DLWKLL$QN 27 730 LSQETF$rSNLWKLL$QN 28 731 LSQQTF$r8NLWKLL$QN29 732 LSQQTF$r8NLWRLL$QN 30 733 QSQQTF$r8NLWKLL$QN 31 734QSQQTF$r8NLWRLL$QN 32 735 QSQQTA$r8NLWRLL$QN 33 736 QSQQTF$F8NLWRKK$QN34 737 QQTF$r8DLWRLL$EN 35 738 QQTF$r8DLWRLL$ 36 739 LSQQTF$DLW$LL 37740 QQTF$DLW$LL 38 741 QQTA$r8DLWRLL$EN 39 742 QSQQTF$r5NLWRLL$s8QN(dihydroxylated olefin) 40 743 QSQQTA$r5NLWRLL$s8QN(dihydroxylated olefin) 41 744 QSQQTF$r8DLWRLL$QN 42 745 QTF$r8NLWRLL$43 746 QSQQTF$NLW$LLPQN 44 747 QS$QTF$NLWRLLPQN 45 748 $TFS$LWKLL 46 749ETF$DLWSLL 47 750 QTF$NLWSLL 48 751 $SQE$FSNLWKLL

In Table 2a, X represents S or any amino acid. Peptides shown cancomprise an N-terminal capping group such as acetyl or an additionallinker such as beta-alanine between the capping group and the start ofthe peptide sequence.

In some embodiments, peptidomimetic macrocycles do not comprise apeptidomimetic macrocycle structure as shown in Table 2a.

In other embodiments, peptidomimetic macrocycles exclude peptidomimeticmacrocycles shown in Table 2b:

TABLE 2b Observed SEQ ID Exact mass Number NO: Sequence Mass M + 2 (m/e)1 752 Ac-LSQETF$r8DLWKLL$EN-NH2 2068.13 1035.07 1035.36 2 753Ac-LSQETF$r8NLWKLL$QN-NH2 2066.16 1034.08 1034.31 3 754Ac-LSQQTF$r8NLWRLL$QN-NH2 2093.18 1047.59 1047.73 4 755Ac-QSQQTF$r8NLWKLL$QN-NH2 2080.15 1041.08 1041.31 5 756Ac-QSQQTF$r8NLWRLL$QN-NH2 2108.15 1055.08 1055.32 6 757Ac-QSQQTA$r8NLWRLL$QN-NH2 2032.12 1017.06 1017.24 7 758Ac-QAibQQTF$r8NLWRLL$QN-NH2 2106.17 1054.09 1054.34 8 759Ac-QSQQTFSNLWRLLPQN-NH2 2000.02 1001.01 1001.26 9 760Ac-QSQQTF$/r8NLWRLL$/QN-NH2 2136.18 1069.09 1069.37 10 761Ac-QSQAibTF$r8NLWRLL$QN-NH2 2065.15 1033.58 1033.71 11 762Ac-QSQQTF$r8NLWRLL$AN-NH2 2051.13 1026.57 1026.70 12 763Ac-ASQQTF$r8NLWRLL$QN-NH2 2051.13 1026.57 1026.90 13 764Ac-QSQQTF$r8ALWRLL$QN-NH2 2065.15 1033.58 1033.41 14 765Ac-QSQETF$r8NLWRLL$QN-NH2 2109.14 1055.57 1055.70 15 766Ac-RSQQTF$r8NLWRLL$QN-NH2 2136.20 1069.10 1069.17 16 767Ac-RSQQTF$r8NLWRLL$EN-NH2 2137.18 1069.59 1069.75 17 768Ac-LSQETFSDLWKLLPEN-NH2 1959.99 981.00 981.24 18 769Ac-QSQ$TFS$LWRLLPQN-NH2 2008.09 1005.05 1004.97 19 770Ac-QSQQ$FSN$WRLLPQN-NH2 2036.06 1019.03 1018.86 20 771Ac-QSQQT$SNL$RLLPQN-NH2 1917.04 959.52 959.32 21 772Ac-QSQQTF$NLW$LLPQN-NH2 2007.06 1004.53 1004.97 22 773Ac-RTQATF$r8NQWAibANle$TNAibTR-NH2 2310.26 1156.13 1156.52 23 774Ac-QSQQTF$r8NLWRLL$RN-NH2 2136.20 1069.10 1068.94 24 775Ac-QSQRTF$r8NLWRLL$QN-NH2 2136.20 1069.10 1068.94 25 776Ac-QSQQTF$r8NNleWRLL$QN-NH2 2108.15 1055.08 1055.44 26 777Ac-QSQQTF$r8NLWRNleL$QN-NH2 2108.15 1055.08 1055.84 27 778Ac-QSQQTF$r8NLWRLNle$QN-NH2 2108.15 1055.08 1055.12 28 779Ac-QSQQTY$r8NLWRLL$QN-NH2 2124.15 1063.08 1062.92 29 780Ac-RAibQQTF$r8NLWRLL$QN-NH2 2134.22 1068.11 1068.65 30 781Ac-MPRFMDYWEGLN-NH2 1598.70 800.35 800.45 31 782Ac-RSQQRF$r8NLWRLL$QN-NH2 2191.25 1096.63 1096.83 32 783Ac-QSQQRF$r8NLWRLL$QN-NH2 2163.21 1082.61 1082.87 33 784Ac-RAibQQRF$r8NLWRLL$QN-NH2 2189.27 1095.64 1096.37 34 785Ac-RSQQRF$r8NFWRLL$QN-NH2 2225.23 1113.62 1114.37 35 786Ac-RSQQRF$r8NYWRLL$QN-NH2 2241.23 1121.62 1122.37 36 787Ac-RSQQTF$r8NLWQLL$QN-NH2 2108.15 1055.08 1055.29 37 788Ac-QSQQTF$r8NLWQAmIL$QN-NH2 2094.13 1048.07 1048.32 38 789Ac-QSQQTF$r8NAmlWRLL$QN-NH2 2122.17 1062.09 1062.35 39 790Ac-NlePRF$r8DYWEGL$QN-NH2 1869.98 935.99 936.20 40 791Ac-NlePRF$r8NYWRLL$QN-NH2 1952.12 977.06 977.35 41 792Ac-RF$r8NLWRLLSQ-NH2 1577.96 789.98 790.18 42 793Ac-QSQQTF$r8N2ffWRLL$QN-NH2 2160.13 1081.07 1081.40 43 794Ac-QSQQTF$r8N3ffWRLL$QN-NH2 2160.13 1081.07 1081.34 44 795Ac-QSQQTF#r8NLWRLL#QN-NH2 2080.12 1041.06 1041.34 45 796Ac-RSQQTA$r8NLWRLL$QN-NH2 2060.16 1031.08 1031.38 46 797Ac-QSQQTF%r8NLWRLL%QN-NH2 2110.17 1056.09 1056.55 47 798HepQSQ$TFSNLWRLLPQN-NH2 2051.10 1026.55 1026.82 48 799HepQSQ$TF$r8NLWRIL$QN-NH2 2159.23 1080.62 1080.89 49 800Ac-QSQQTF$r8NL6clWRLL$QN-NH2 2142.11 1072.06 1072.35 50 801Ac-QSQQTF$r8NLMe6clwRLL$QN-NH2 2156.13 1079.07 1079.27 51 802Ac-LTFEHYWAQLTS-NH2 1535.74 768.87 768.91 52 803 Ac-LTF$HYW$QLTS-NH21585.83 793.92 794.17 53 804 Ac-LTFE$YWA$LTS-NH2 1520.79 761.40 761.6754 805 Ac-LTF$zr8HYWAQL$zS-NH2 1597.87 799.94 800.06 55 806Ac-LTF$r8HYWRQL$S-NH2 1682.93 842.47 842.72 56 807Ac-QS$QTFStNLWRLL$s8QN-NH2 2145.21 1073.61 1073.90 57 808Ac-QSQQTASNLWRLLPQN-NH2 1923.99 963.00 963.26 58 809Ac-QSQQTA$/r8NLWRLL$/QN-NH2 2060.15 1031.08 1031.24 59 810Ac-ASQQTF$/r8NLWRLL$/QN-NH2 2079.16 1040.58 1040.89 60 811Ac-$SQQ$FSNLWRLLAibQN-NH2 2009.09 1005.55 1005.86 61 812Ac-QS$QTF$NLWRLLAibQN-NH2 2023.10 1012.55 1012.79 62 813Ac-QSQQ$FSN$WRLLAibQN-NH2 2024.06 1013.03 1013.31 63 814Ac-QSQQTF$NLWSLLAibQN-NH2 1995.06 998.53 998.87 64 815Ac-QSQQTFS$LWRSLAibQN-NH2 2011.06 1006.53 1006.83 65 816Ac-QSQQTFSNLW$LLA$N-NH2 1940.02 971.01 971.29 66 817Ac-$/SQQ$/FSNLWRLLAibQN-NH2 2037.12 1019.56 1019.78 67 818Ac-QS$/QTF$/NLWRLLAibQN-NH2 2051.13 1026.57 1026.90 68 819Ac-QSQQ$/FSN$/WRLLAibQN-NH2 2052.09 1027.05 1027.36 69 820Ac-QSQQTF$/NLW$/LLAibQN-NH2 2023.09 1012.55 1013.82 70 821Ac-QSQ$TFS$LWRLLAibQN-NH2 1996.09 999.05 999.39 71 822Ac-QSQ$/TFS$/LWRLLAibQN-NH2 2024.12 1013.06 1013.37 72 823Ac-QS$/QTFSt//NLWRLL$/s8QN-NH2 2201.27 1101.64 1102.00 73 824Ac-$r8SQQTFS$LWRLLAibQN-NH2 2038.14 1020.07 1020.23 74 825Ac-QSQ$r8TFSNLW$LLAibQN-NH2 1996.08 999.04 999.32 75 826Ac-QSQQTFS$r8LWRLLA$N-NH2 2024.12 1013.06 1013.37 76 827Ac-QS$r5QTFStNLW$LLAibQN-NH2 2032.12 1017.06 1017.39 77 828Ac-$/r8SQQTFS$/LWRLLAibQN-NH2 2066.17 1034.09 1034.80 78 829Ac-QSQ$/r8TFSNLW$/LLAibQN-NH2 2024.11 1013.06 1014.34 79 830Ac-QSQQTFS$/r8LWRLLA$/N-NH2 2052.15 1027.08 1027.16 80 831Ac-QS$/r5QTFSt//NLW$/LLAibQN-NH2 2088.18 1045.09 1047.10 81 832Ac-QSQQTFSNLWRLLAibQN-NH2 1988.02 995.01 995.31 82 833Hep/QSQ$/TF$/r8NLWRLL$/QN-NH2 2215.29 1108.65 1108.93 83 834Ac-ASQQTF$rNLRWLL$QN-NH2 2051.13 1026.57 1026.90 84 835Ac-QSQQTF$/r8NLWRLL$/Q-NH2 2022.14 1012.07 1012.66 85 836Ac-QSQQTF$r8NLWRLL$Q-NH2 1994.11 998.06 998.42 86 837Ac-AAARAA$r8AAARAA$AA-NH2 1515.90 758.95 759.21 87 838Ac-LTFEHYWAQLTSA-NH2 1606.78 804.39 804.59 88 839 Ac-LTF$r8HYWAQL$$A-NH21668.90 835.45 835.67 89 840 Ac-ASQQTFSNLWRLLPQN-NH2 1943.00 972.50973.27 90 841 Ac-QS$QTFStNLW$r5LLAibQN-NH2 2032.12 1017.06 1017.30 91842 Ac-QSQQTFAibNLWRLLAibQN-NH2 1986.04 994.02 994.19 92 843Ac-QSQQTFNleNLWRLLNleQN-NH2 2042.11 1022.06 1022.23 93 844Ac-QSQQTF$/r8NLWRLLAibQN-NH2 2082.14 1042.07 1042.23 94 845Ac-QSQQTF$/r8NLWRLLNleQN-NH2 2110.17 1056.09 1056.29 95 846Ac-QSQQTFAibNLWRLL$/QN-NH2 2040.09 1021.05 1021.25 96 847Ac-QSQQTFNleNLWRLL$/QN-NH2 2068.12 1035.06 1035.31 97 848Ac-QSQQTF%r8NL6clWRNleL%QN-NH2 2144.13 1073.07 1073.32 98 849Ac-QSQQTF%r8NLMe6clWRLL%QN-NH2 2158.15 1080.08 1080.31 101 850Ac-FNle$YWE$L-NH2 1160.63 — 1161.70 102 851 Ac-F$r8AYWELL$A-NH2 1344.75— 1345.90 103 852 Ac-F$r8AYWQLL$A-NH2 1343.76 — 1344.83 104 853Ac-NlePRF$r8NYWELL$QN-NH2 1925.06 963.53 963.69 105 854Ac-NlePRF$r8DYWRLL$QN-NH2 1953.10 977.55 977.68 106 855Ac-NlePRF$8NYWRLL$Q-NH2 1838.07 920.04 920.18 107 856Ac-NlePRF$r8NYWRLL$-NH2 1710.01 856.01 856.13 108 857Ac-QSQQTF$r8DLWRLL$QN-NH2 2109.14 1055.57 1055.64 109 858Ac-QSQQTF$r8NLWRLL$EN-NH2 2109.14 1055.57 1055.70 110 859Ac-QSQQTF$r8NLWRLL$QD-NH2 2109.14 1055.57 1055.64 111 860Ac-QSQQTF$r8NLWRLL$S-NH2 1953.08 977.54 977.60 112 861Ac-ESQQTF$r8NLWRLL$QN-NH2 2109.14 1055.57 1055.70 113 862Ac-LTF$r8NLWRNleL$Q-NH2 1635.99 819.00 819.10 114 863Ac-LRF$r8NLWRNleL$Q-NH2 1691.04 846.52 846.68 115 864Ac-QSQQTF$r8NWWRNleL$QN-NH2 2181.15 1091.58 1091.64 116 865Ac-QSQQTF$r8NLWRNleL$Q-NH2 1994.11 998.06 998.07 117 866Ac-QTF$r8NLWRNleL$QN-NH2 1765.00 883.50 883.59 118 867Ac-NlePRF$r8NWWRLL$QN-NH 1975.13 988.57 988.75 119 868Ac-NlePRF$r8NWWRLL$A-NH2 1804.07 903.04 903.08 120 869 Ac-TSFAEYWNLLNH21467.70 734.85 734.90 121 870 Ac-QTF$r8HWWSQL$S-NH2 1651.85 826.93827.12 122 871 Ac-FM$YWE$L-NH2 1178.58 — 1179.64 123 872Ac-QTFEHWWSQLLS-NH2 1601.76 801.88 801.94 124 873Ac-QSQQTF$r8NLAmwRLNle$QN-NH2 2122.17 1062.09 1062.24 125 874Ac-FMAibY6clWEAc3cL-NH2 1130.47 — 1131.53 126 875 Ac-FNle$Y6clWE$L-NH21194.59 — 1195.64 127 876 Ac-F$zr8AY6clWEAc3cL$z-NH2 1277.63 639.821278.71 128 877 Ac-F$r8AY6clWEAc3cL$A-NH2 1348.66 — 1350.72 129 878Ac-NlePRF$r8NY6clWRLL$QN-NH2 1986.08 994.04 994.64 130 879Ac-AF$r8AAWALA$A-NH2 1223.71 — 1224.71 131 880 Ac-TF$rAAWRLA$Q-NH21395.80 698.90 399.04 132 881 Pr-TF$r8AAWRLA$Q-NH2 1409.82 705.91 706.04133 882 Ac-QSQQTF%r8NLWRNleL%QN-NH2 2110.17 1056.09 1056.22 134 883Ac-LTF%r8HYWAQL%$A-NH2 1670.92 836.46 836.58 135 884Ac-NlePRF%r8NYWRLL%QN-NH2 1954.13 978.07 978.19 136 885Ac-NlePRF%r8NY6clWRLL%QN-NH2 1988.09 995.05 995.68 137 886Ac-LTF%r8HY6clWAQL%S-NH2 1633.84 817.92 817.93 138 887Ac-QS%QTF%StNLWRLL%s8QN-NH2 2149.24 1075.62 1075.65 139 888Ac-LTF%r8HY6clWRQL%S-NH2 1718.91 860.46 860.54 140 889Ac-QSQQTF%r8NL6clWRLL%QN-NH2 2144.13 1073.07 1073.64 141 890Ac-%r8SQQTFS%LWRLLAibQN-NH2 2040.15 1021.08 1021.13 142 891Ac-LTF%r8HYWAQL%S-NH2 1599.88 800.94 801.09 143 892Ac-TSF%r8QYWNLL%P-NH2 1602.88 802.44 802.58 147 893 Ac-LTFEHYWAQLTS-NH21535.74 768.87 769.5 152 894 Ac-F$er8AY6clWEAc3cL$e-NH2 1277.63 639.821278.71 153 895 Ac-AF$r8AAWALA$A-NH2 1277.63 639.82 1277.84 154 896Ac-TF$r8AAWRLA$Q-NH2 1395.80 698.90 699.04 155 897 Pr-TF$r8AAWRLA$Q-NH21409.82 705.91 706.04 156 898 Ac-LTF$er8HYWAQL$eS-NH2 1597.87 799.94800.44 159 899 Ac-CCPGCCBaQSQQTF$r8NLWRLL$QN-NH2 2745.30 1373.65 1372.99160 900 Ac-CCPGCCBaQSQQTA$r8NLWRLL$QN-NH2 2669.27 1335.64 1336.09 161901 Ac-CCPGCCBaNlePRF$r8NYWRLL$QN-NH2 2589.26 1295.63 1296.2 162 902Ac-LTF$/r8HYWAQL$/S-NH2 1625.90 813.95 814.18 163 903Ac-F%r8HY6clWRAc3cL%-NH2 1372.72 687.36 687.59 164 904Ac-QTF%r8HWWSQL%S-NH2 1653.87 827.94 827.94 165 905Ac-LTA$r8HYWRQL$S-NH2 1606.90 804.45 804.66 166 906Ac-Q$r8QQTFSN$WRLLAibQN-NH2 2080.12 1041.06 1041.61 167 907Ac-QSQQ$r8FSNLWR$LAibQN-NH2 2066.11 1034.06 1034.58 168 908Ac-F$r8AYWEAc3cL$A-NH2 1314.70 658.35 1315.88 169 909Ac-F$r8AYWEAc3cL$S-NH2 1330.70 666.35 1331.87 170 910Ac-F$r8AYWEAc3cL$Q-NH2 1371.72 686.86 1372.72 171 911Ac-F$r8AYWEAibL$S-NH2 1332.71 667.36 1334.83 172 912 Ac-F$r8AYWEAL$S-NH21318.70 660.35 1319.73 173 913 Ac-F$f8AYWEQL$S-NH2 1375.72 688.861377.53 174 914 Ac-F$r8HYWEQL$S-NH2 1441.74 721.87 1443.48 175 915Ac-F$r8HYWAQL$S-NH2 1383.73 692.87 1385.38 176 916Ac-F$r8HYWAAc3cL$S-NH2 1338.71 670.36 1340.82 177 917Ac-F$r8HYWRAc3cL$S-NH2 1423.78 712.89 713.04 178 918Ac-F$r8AYWEAc3cL#A-NH2 1300.69 651.35 1302.78 179 919Ac-NlePTF%r8NYWRLL%QN-NH2 1899.08 950.54 950.56 180 920Ac-TF$r8AAWRAL$Q-NH2 1395.80 698.90 699.13 181 921 Ac-TSF%r8HYWAQL%S-NH21573.83 787.92 787.98 184 922 Ac-F%r8AY6clWEAc3cL%A-NH2 1350.68 676.34676.91 185 923 Ac-LTF$r8HYWAQI$S-NH2 1597.87 799.94 800.07 186 924Ac-LTF$r8HYWAQNle$S-NH2 1597.87 799.94 800.07 187 925Ac-LTF$r8HYWAQL$A-NH2 1581.87 791.94 792.45 188 926Ac-LTF$r8HYWAQL$Abu-NH2 1595.89 798.95 799.03 189 927Ac-LTF$r8HYWAbuQL$S-NH2 1611.88 806.94 807.47 190 928Ac-LTF$er8AYWAQL$eS-NH2 1531.84 766.92 766.96 191 929Ac-LAF$rHYWAQL$S-NH 1567.86 784.93 785.49 192 930 Ac-LAF$rAYWAQL$S-NH1501.83 751.92 752.01 193 931 Ac-LTF$er8AYWAQL$eA-NH2 1515.85 758.93758.97 194 932 Ac-LAF$r8AYWAQL$A-NH2 1485.84 743.92 744.05 195 933Ac-LTF$8NLWANleL$Q-NH2 1550.92 776.46 776.61 196 934Ac-LTF$r8NLWANleL$A-NH2 1493.90 747.95 1495.6 197 935Ac-LTF$r8ALWANleL$Q-NH2 1507.92 754.96 755 198 936Ac-LAF$r8NIWANicL$Q-NH2 1520.91 761.46 761.96 199 937Ac-LAF$r8ALWANleL$A-NH2 1420.89 711.45 1421.74 200 938Ac-A$r8AYWEAc3cL$A-NH2 1238.67 620.34 1239.65 201 939Ac-F$r8AYWEAc3cL$AA-NH2 1385.74 693.87 1386.64 202 940Ac-F$r8AYWEAc3cL$AbU-NH2 1328.72 665.36 1330.17 203 941Ac-T$r8AYWEAc3cL$Nle-NH2 1356.75 679.38 1358.22 204 942Ac-F$r5AYWEAc3cL$s8A-NH2 1314.70 658.35 1315.51 205 943Ac-F$AYWEAc3cL$r8A-NH2 1314.70 658.35 1315.66 206 944Ac-F$r8AYWEAc3cI$A-NH2 1314.70 658.35 1316.18 207 945Ac-F$r8AYWEAc3cNie$A-NH2 1314.70 658.35 1315.66 208 946Ac-F$r8AYWEAmIL$A-NH2 1358.76 680.38 1360.21 209 947Ac-FSrAYWENlel$A-NH2 1344.75 673.38 1345.71 210 948Ac-F$r8AYWQAc3cL$A-NH2 1313.72 657.86 1314.7 211 949Ac-F$r8AYWAAc3cL$A-NH2 1256.70 629.35 1257.56 212 950Ac-F$r8AYWAbUAc3cL$A-NH2 1270.71 636.36 1272.14 213 951Ac-F$r8AYWNleAc3cL$A-NH2 1298.74 650.37 1299.67 214 952Ac-F$r8AbuYWEAc3cL$A-NH2 1328.72 665.36 1329.65 215 953Ac-F$r8NleYWEAc3cL$A-NH2 1356.75 679.38 1358.66 216 9545-FAM-BaLTFEHYWAQLTS-NH2 1922.82 962.41 962.87 217 9555-FAM-BaLTF%r8HYWAQL%S-NH2 1986.96 994.48 994.97 218 956Ac-LTF$r8HYWAQhL$S-NH2 1611.88 806.94 807 219 957Ac-LTF$r8HYWAQTle$S-NH2 1597.87 799.94 799.97 220 958Ac-LTF$r8HYWAQAdm$S-NH2 1675.91 838.96 839.09 221 959Ac-LTF$r8HYWAQhCha$S-NH2 1651.91 826.96 826.98 222 960Ac-LTF$r8HYWAQCha$S-NH2 1637.90 819.95 820.02 223 961Ac-LTF$r8HYWAc6cQL$S-NH2 1651.91 826.96 826.98 224 962Ac-LTF$r8HYWAC5cQL$S-NH2 1637.90 819.95 820.02 225 963Ac-LThF$r8HYWAQL$S-NH2 1611.88 806.94 807 226 964Ac-LTIgl$r8HYWAQL$S-NH2 1625.90 813.95 812.99 227 965Ac-LTF$r8HYWAQChg$S-NH2 1623.88 812.94 812.99 228 966Ac-LTF$r8HYWAQF$S-NH2 1631.85 816.93 816.99 229 967Ac-LTF$r8HYWAQIgI$S-NH2 1659.88 830.94 829.94 230 968Ac-LTF$r8HYWAQCba$S-NH2 1609.87 805.94 805.96 231 969Ac-LTF$r8HYWAQCpg$S-NH2 1609.87 805.94 805.96 232 970Ac-LTF$8HhYWAQL$S-NH2 1611.88 806.94 807 233 971 Ac-F$r8AYWEAc3chL$A-NH21328.72 665.36 665.43 234 972 Ac-F$r8AYWEAc3cTle$A-NH2 1314.70 658.351315.62 235 973 Ac-F$r8AYWEAc3cAdm$A-NH2 1392.75 697.38 697.47 236 974Ac-F$r8AYWEAc3chCha$A-NH2 1368.75 685.38 685.34 237 975Ac-F$r8AYWEAc3cCha$A-NH2 1354.73 678.37 678.38 238 976Ac-F$r8AYWEACGCL$A-NH2 1356.75 679.38 679.42 239 977Ac-F$r8AYWEACSCL$A-NH2 1342.73 672.37 672.46 240 978Ac-hF$r8AYWEAc3cL$A-NH2 1328.72 665.36 665.43 241 970Ac-Igl$r8AWEAc3cL$A-NH2 1342.73 672.37 671.5 243 980Ac-F$r8AYWEAc3cF$A-NH2 1348.69 675.35 675.35 244 981Ac-F$r8AYWEAc3cIgl$A-NH2 1376.72 689.36 688.37 245 982Ac-F$r8AYWEAc3cCba$A-NH2 1326.70 664.35 664.47 246 983Ac-F$r8AYWEAc3cCpg$A-NH2 1326.70 664.35 664.39 247 984Ac-F$r8AhYWEAc3cL$A-NH2 1328.72 665.36 665.43 248 985Ac-F$r8AYWEAc3cL$Q-NH2 1371.72 686.86 1372.87 249 986Ac-F$r8AYWEAibL$A-NH2 1316.72 659.36 1318.18 250 987 Ac-F$r8AYWEAL$A-NH21302.70 652.35 1303.75 251 988 Ac-LAF$r8AYWAAL$A-NH2 1428.82 715.41715.49 252 989 Ac-LTF$r8HYWAAc3cL$S-NH2 1552.84 777.42 777.5 253 990Ac-NleTF$r8HYWAQL$S-NH2 1597.87 799.94 800.04 254 991Ac-VTF$r8HYWAQL$S-NH2 1583.85 792.93 793.04 255 992Ac-FTF$r8HYWAQL$S-NH2 1631.85 816.93 817.02 256 993Ac-WTF$r8HYWAQL$S-NH2 1670.86 836.43 836.85 257 994 Ac-RTF$rHYWAQL$S-NH1640.88 821.44 821.9 258 995 Ac-KTF$r8HYWAQL$S-NH2 1612.88 807.44 807.91259 996 Ac-LNleF$r8HYWAQL$S-NH2 1609.90 805.95 806.43 260 997Ac-LVF$F8HYWAQL$S-NH2 1595.89 798.95 798.93 261 998Ac-LFF$r8HYWAQL$S-NH2 1643.89 822.95 823.38 262 999Ac-LWF$F8HYWAQL$S-NH2 1682.90 842.45 842.55 263 1000Ac-LRF$r8HYWAQL$S-NH2 1652.92 827.46 827.52 264 1001Ac-LKF$r8HYWAQL$S-NH2 1624.91 813.46 813.51 265 1002Ac-LTF$r8NleYWAQL$S-NH2 1573.89 787.95 788.05 266 1003Ac-LTF$8VYWAQL$S-NH2 1559.88 780.94 780.98 267 1004Ac-LTF$r8FYWAQL$S-NH2 1607.88 804.94 805.32 268 1005Ac-LTF$r8WYWAQL$S-NH2 1646.89 824.45 824.86 269 1006Ac-LTF$8RYWAQL$S-NH2 1616.91 809.46 809.51 270 1007 Ac-LTF$rKYWAQL$S-NH1588.90 795.45 795.48 271 1008 Ac-LTF$r8HNleWAQL$S-NH2 1547.89 774.95774.98 272 1009 Ac-LTF$r8HVWAQL$S-NH2 1533.87 767.94 767.95 273 1010Ac-LTF$8HFWAQL$S-NH2 1581.87 791.94 792.3 274 1011 Ac-LTF$r8HWWAQL$S-NH21620.88 811.44 811.54 275 1012 Ac-LTF$r8HRWAQL$S-NH2 1590.90 796.45796.52 276 1013 Ac-LTF$r8HKWAQL$S-NH2 1562.90 782.45 782.53 277 1014Ac-LTF$r8HYWNleQL$S-NH2 1639.91 820.96 820.98 278 1015Ac-LTF$rHYWVQL$S-NH 1625.90 813.95 814.03 279 1016 Ac-LTF$r8HYWFQL$S-NH21673.90 837.95 838.03 280 1017 Ac-LTF$r8HYWWQL$S-NH2 1712.91 857.46857.5 281 1018 Ac-LTF$r8HYWKQL$S-NH2 1654.92 828.46 828.49 282 1019Ac-LTF$r8HYWANleL$S-NH2 1582.89 792.45 792.52 283 1020Ac-LTF$r8HYWAVL$S-NH 1568.88 785.44 785.49 284 1021Ac-LTF$r8HYWAFL$S-NH2 1616.88 809.44 809.47 285 1022Ac-LTF$r8HYWAWL$S-NH2 1655.89 828.95 829 286 1023 Ac-LTF$r8HYWARL$S-NH21625.91 813.96 813.98 287 1024 Ac-LTF$r8HYWAQL$Nle-NH2 1623.92 812.96813.39 288 1025 Ac-LTF$r8HYWAQL$V-NH2 1609.90 805.95 805.99 289 1026Ac-LTF$rHYWAQL$F-NH 1657.90 829.95 830.26 290 1027 Ac-LTF$r8HYWAQL$W-NH21696.91 849.46 849.5 291 1028 Ac-LTF$r8HYWAQL$R-NH2 1666.94 834.47834.56 292 1029 Ac-LTF$r8HYWAQL$K-NH2 1638.93 820.47 820.49 293 1030Ac-Q$r8QQTFSN$WRLLAibQN-NH2 2080.12 1041.06 1041.54 294 1031Ac-QSQQ$r8FSNLWR$LAibQN-NH2 2066.11 1034.06 1034.58 295 1032Ac-LT2Pal$r8HYWAQL$S-NH2 1598.86 800.43 800.49 296 1033Ac-LT3Pal$r8HYWAQL$S-NH2 1598.86 800.43 800.49 297 1034Ac-LT4Pal$r8HYWAQL$S-NH2 1598.86 800.43 800.49 298 1035Ac-LTF2CF3$r8HYWAQL$S-NH2 1665.85 833.93 834.01 299 1036Ac-LTF2CN$r8HYWAQL$S-NH2 1622.86 812.43 812.47 300 1037Ac-ITF2Me$r8HYWAQL$S-NH2 1611.88 806.94 807 301 1038Ac-LTF3Cl$r8HYWAQL$S-NH2 1631.83 816.92 816.99 302 1039Ac-LTF4CF3$r8HYWAQL$S-NH2 1665.85 833.93 833.94 303 1040Ac-LTF4tBu$r8HYWAQL$S-NH2 1653.93 827.97 828.02 304 1041Ac-LTF5F$r8HYWAQL$S-NH2 1687.82 844.91 844.96 305 1042Ac-LTF$r8HY3BthAAQL$S-NH2 1614.83 808.42 808.48 306 1043Ac-LTF2Br$r8HYWAQL$S-NH2 1675.78 838.89 838.97 307 1044Ac-LTF4Br$rSHYWAQL$S-NH2 1675.78 838.89 839.86 308 1045Ac-LTF2Cl$r8HYWAQL$S-NH2 1631.83 816.92 816.99 309 1046Ac-LTF4Cl$r8HYWAQL$S-NH2 1631.83 816.92 817.36 310 1047Ac-LTF3CN$r8HYWAQL$S-NH2 1622.86 812.43 812.47 311 1048Ac-LTFACN$r8HYWAQL$S-NH2 1622.86 812.43 812.47 312 1049Ac-LTF34C12$r8HYWAQL$S-NH2 1665.79 833.90 833.94 313 1050Ac-LTF34F2$r8HYWAQI$S-NH2 1633.85 817.93 817.95 314 1051Ac-LTF35F2$r8HYWAQL$S-NH2 1633.85 817.93 817.95 315 1052Ac-LTDip$r8HYWAQL$S-NH2 1673.90 837.95 838.01 316 1053Ac-LTF2F$r8HYWAQL$S-NH2 1615.86 808.93 809 317 1054Ac-LTF3F$r8HYWAQL$S-NH2 1615.86 808.93 809 318 1055Ac-LTF4F$r8HYWAQL$S-NH2 1615.86 808.93 809 319 1056Ac-LTF4I$r8HYWAQL$S-NH2 1723.76 862.88 862.94 320 1057Ac-LTF3Me$r8HYWAQL$S-NH2 1611.88 806.94 807.07 321 1058Ac-LTF4Me$r8HYWAQL$S-NH2 1611.88 806.94 807 322 1059Ac-LT1Nal$r8HYWAQL$S-NH2 1647.88 824.94 824.98 323 1060Ac-LT2Nal$r8HYWAQL$S-NH2 1647.88 824.94 825.06 324 1061Ac-LTF3CF3$r8HYWAQL$S-NH2 1665.85 833.93 834.01 325 1062Ac-LTF4NO2$r8T-mVAQL$S-NH2 1642.85 822.43 822.46 326 1063Ac-LTF3NO2$r8HYWAQL$S-NH2 1642.85 822.43 822.46 327 1064Ac-LTF$r82ThiYWAQL$S-NH2 1613.83 807.92 807.96 328 1065Ac-LTF$r8HBipWAQL$S-NH2 1657.90 829.95 830.01 329 1066Ac-LTF$r8HF4tBuWAQL$S-NH2 1637.93 819.97 820.02 330 1067Ac-LTF$r8HF4CF3WAQL$S-NH2 1649.86 825.93 826.02 331 1068Ac-LTF$rHF4ClWAQL$S-NH2 1615.83 808.92 809.37 332 1069Ac-LTF$8HF4MeWAQL$S-NH2 1595.89 798.95 799.01 333 1070Ac-LTF$r8HF4BrWAQL$S-NH2 1659.78 830.89 830.98 334 1071Ac-LTF$r8HF4CNWAQL$S-NH2 1606.87 804.44 804.56 335 1072Ac-LTF$8HF4NO2WAQL$S-NH2 1626.86 814.43 814.55 336 1073Ac-LTF$r8H1NalWAQL$S-NH2 1631.89 816.95 817.06 337 1074Ac-LTF$r8H2NalWAQL$S-NH2 1631.89 816.95 816.99 338 1075Ac-LTF$r8HWAQL$S-NH2 1434.80 718.40 718.49 339 1076Ac-LTF$r8HYINalAQL$S-NH2 1608.87 805.44 805.52 340 1077Ac-LTF$r8HY2NalAQL$S-NH2 1608.87 805.44 805.52 341 1078Ac-LTF$r8HYWAQI$S-NH2 1597.87 799.94 800.07 342 1079Ac-ITF$r8HYWAQNle$S-NH2 1597.87 799.94 800.44 343 1080Ac-LTF$er8HYWAQL$eA-NH2 1581.87 791.94 791.98 344 1081Ac-LTF$r8HYWAQL$AbU-NH2 1595.89 798.95 799.03 345 1082Ac-LTF$r8HYWAbUQL$S-NH2 1611.88 806.94 804.47 346 1083Ac-LAF$r8HYWAQL$S-NH2 1567.86 784.93 785.49 347 1084Ac-LTF$r8NLWANleL$Q-NH2 1550.92 776.46 777.5 348 1085Ac-LTF$r8ALWANleL$Q-NH2 1507.92 754.96 755.52 349 1086Ac-LAF$r8NLWANleL$Q-NH2 1520.91 761.46 762.48 350 1087Ac-F$r8AYWAAc3cL$A-NH2 1256.70 629.35 1257.56 351 1088Ac-LTF$r8AYWAAL$S-NH2 1474.82 738.41 738.55 352 1089Ac-LVF$r8AYWAQL$S-NH2 1529.87 765.94 766 353 1090 Ac-LTF$rAYWAbUQL$S-NH21545.86 773.93 773.92 354 1091 Ac-LTF$8AYWNleQL$S-NH2 1573.89 787.95788.17 355 1092 Ac-LTF$r8AbuYWAQL$S-NH2 1545.86 773.93 773.99 356 1093Ac-LTF$r8AYWHQL$S-NH2 1597.87 799.94 799.97 357 1094Ac-LTF$8AYWKQL$S-NH2 1588.90 795.45 795.53 358 1095Ac-LTF$r8AYWOQL$S-NH2 1574.89 788.45 788.5 359 1096Ac-LTF$r8AYWRQL$S-NH2 1616.91 809.46 809.51 360 1097Ac-LTF$rAYWSQL$S-NH2 1547.84 774.92 774.96 361 1098 Ac-LTF$AYWRAL$S-NH21559.89 780.95 780.95 362 1099 Ac-LTF$r8AYWRQL$A-NH2 1600.91 801.46801.52 363 1100 Ac-LTF$r8AYWRAL$A-NH2 1543.89 772.95 773.03 364 1101Ac-LTF$r5HYWAQL$s8S-NH2 1597.87 799.94 799.97 365 1102Ac-LTF$HYWAQL$r8S-NH2 1597.87 799.94 799.97 366 1103Ac-LTF$r8HYWAAL$S-NH2 1540.84 771.42 771.48 367 1104Ac-LTF$r8HYWAAbLL$S-NH2 1554.86 778.43 778.51 368 1105Ac-LTF$r8HYWALL$S-NH2 1582.89 792.45 792.49 369 1106 Ac-F$r8AYWHAL$A-NH21310.72 656.36 656.4 370 1107 Ac-F$r8AYWAAL$A-NH2 1244.70 623.35 1245.61371 1108 Ac-F$rAYWSAL$A-NH2 1260.69 631.35 1261.6 372 1109Ac-F$r8AYWRAL$A-NH2 1329.76 665.88 1330.72 373 1110 Ac-F$r8AYWKAL$A-NH21301.75 651.88 1302.67 374 1111 Ac-F$r8AYWOAL$A-NH2 1287.74 644.871289.13 375 1112 Ac-F$r8VYWEAc3cL$A-NH2 1342.73 672.37 1343.67 376 1113Ac-F$r8FYWEAc3cL$A-NH2 1390.73 696.37 1392.14 377 1114Ac-F$r8WYWEAc3cL$A-NH2 1429.74 715.87 1431.44 378 1115Ac-F$r8RYWEAc3cL$A-NH2 1399.77 700.89 700.95 379 1116Ac-F$r8KYWEAc3cL$A-NH2 1371.76 686.88 686.97 380 1117Ac-F$r8ANleWEAc3cL$A-NH2 1264.72 633.36 1265.59 381 1118Ac-F$r8AVWEAc3cL$A-NH2 1250.71 626.36 1252.2 382 1119Ac-F$r8AFWEAc3cL$A-NH2 1298.71 650.36 1299.64 383 1120Ac-F$r8AWWEAc3cL$A-NH2 1337.72 669.86 1338.64 384 1121Ac-F$r8ARWEAc3cL$A-NH2 1307.74 654.87 655 385 1122Ac-F$r8AKWEAc3cL$A-NH2 1279.73 640.87 641.01 386 1123Ac-F$r8AYWVAc3cL$A-NH2 1284.73 643.37 643.38 387 1124Ac-F$r8AYWFAc3cL$A-NH2 1332.73 667.37 667.43 388 1125Ac-F$r8AYWWAc3cL$A-NH2 1371.74 686.87 686.97 389 1126Ac-F$rAYWRAc3cL$A-NH2 1341.76 671.88 671.94 390 1127Ac-F$AYWKAc3cL$A-NH2 1313.75 657.88 657.88 391 1128 Ac-F$r8AYWEVL$A-NH21330.73 666.37 666.47 392 1129 Ac-F$r8AYWEFL$A-NH2 1378.73 690.37 690.44393 1130 Ac-F$r8AYWEWL$A-NH2 1417.74 709.87 709.91 394 1131Ac-F$r8AYWERL$A-NH2 1387.77 694.89 1388.66 395 1132 Ac-F$r8AYWEKL$A-NH21359.76 680.88 1361.21 396 1133 Ac-F$r8AYWEAc3cL$V-NH2 1342.73 672.371343.59 397 1134 Ac-F$r8AYWEAc3cL$F-NH2 1390.73 696.37 1392.58 398 1135Ac-F$r8AYWEAc3cL$W-NH2 1429.74 715.87 1431.29 399 1136Ac-F$r8AYWEAc3cL$R-NH2 1399.77 700.89 700.95 400 1137Ac-F$r8AYWEAc3cL$K-NH2 1371.76 686.88 686.97 401 1138Ac-F$r8AYWEAc3cL$AV-NH2 1413.77 707.89 707.91 402 1139Ac-F$r8AYWEAc3cL$AF-NH2 1461.77 731.89 731.96 403 1140Ac-F$r8AYWEAc3cL$AW-NH2 1500.78 751.39 751.5 404 1141Ac-F$r8AYWEAc3cL$AR-NH2 1470.80 736.40 736.47 405 1142Ac-F$r8AYWEAc3cL$AK-NH2 1442.80 722.40 722.41 406 1143Ac-F$r8AYWEAc3cL$AH-NH2 1451.76 726.88 726.93 407 1144Ac-LTF2NO2$r8HYWAQL$S-NH2 1642.85 822.43 822.54 408 1145Ac-LTA$r8HYAAQL$S-NH2 1406.79 704.10 704.5 409 1146Ac-LTF$r8HYAAQL$S-NH2 1482.82 742.41 742.47 410 1147Ac-QSQQTF$r8NLWALL$AN-NH2 1966.07 984.04 984.38 411 1148Ac-QAibQQTF$r8NLWALL$AN-NH2 1964.09 983.05 983.42 412 1149Ac-QAibQQTF$r8ALWALL$AN-NH2 1921.08 961.54 961.59 413 1150Ac-AAAATF$r8AAWAAL$AA-NH2 1608.90 805.45 805.52 414 1151Ac-F$r8AAWRAL$Q-NH2 1294.76 648.38 648.48 415 1152 Ac-TF$r8AAWAAL$Q-NH21310.74 656.37 1311.62 416 1153 Ac-TF$r8AAWRAL$A-NH2 1338.78 670.39670.46 417 1154 Ac-VF$r8AAWRAL$Q-NH2 1393.82 697.91 697.99 418 1155Ac-AF$r8AAWAAL$A-NH2 1223.71 612.86 1224.67 420 1156Ac-TF$r8AAWKAL$Q-NH2 1367.80 684.90 684.97 421 1157 Ac-TF$r8AAWOAL$Q-NH21353.78 677.89 678.01 422 1158 Ac-TF$r8AAWSAL$Q-NH2 1326.73 664.37664.47 423 1159 Ac-LTF$r8AAWRAL$Q-NH2 1508.89 755.45 755.49 424 1160Ac-F$f8AYWAQL$A-NH2 1301.72 651.86 651.96 425 1161 Ac-F$r8AWWAAL$A-NH21267.71 634.86 634.87 426 1162 Ac-F$r8AWWAQL$A-NH2 1324.73 663.37 663.43427 1163 Ac-F$r8AYWEAL$-NH2 1231.66 616.83 1232.93 428 1164Ac-F$r8AYWAAL$-NH2 1173.66 587.83 1175.09 429 1165 Ac-F$r8AYWKAL$-NH21230.72 616.36 616.44 430 1166 Ac-F$r8AYWOAL$-NH2 1216.70 609.35 609.48431 1167 Ac-F$rAYWQAL$-NH2 1230.68 616.34 616.44 432 1168Ac-F$r8AYWAQL$-NH2 1230.68 616.34 616.37 433 1169 Ac-F$r8HYWDQL$S-NH21427.72 714.86 714.86 434 1170 Ac-F$r8HFWEQL$S-NH2 1425.74 713.87 713.98435 1171 Ac-F$r8AYWHQL$S-NH2 1383.73 692.87 692.96 436 1172Ac-F$r8AYWKQL$S-NH2 1374.77 688.39 688.45 437 1173 Ac-F$r8AYWOQL$S-NH21360.75 681.38 681.49 438 1174 Ac-F$r8HYWSQL$S-NH2 1399.73 700.87 700.95439 1175 Ac-F$r8HWWEQL$S-NH2 1464.76 733.38 733.44 440 1176Ac-F$r8HWWAQL$S-NH2 1406.75 704.38 704.43 441 1177 Ac-F$r8AWWHQL$S-NH21406.75 704.38 704.43 442 1178 Ac-F$r8AWWKQL$S-NH2 1397.79 699.90 699.92443 1179 Ac-F$r8AWWOQL$S-NH2 1383.77 692.89 692.96 444 1180Ac-F$r8HWWSQI$S-NH2 1422.75 712.38 712.42 445 1181Ac-LTF$r8NYWANleL$Q-NH2 1600.90 801.45 801.52 446 1182Ac-LTF$8NLWAQL$Q-NH2 1565.90 783.95 784.06 447 1183Ac-LTF$r8NYWANleL$A-NH2 1543.88 772.94 773.03 448 1184Ac-LTF$r8NLWAQI$A-NH2 1508.88 755.44 755.49 449 1185Ac-LTF$r8AYWANleL$Q-NH2 1557.90 779.95 780.06 450 1186Ac-LTF$8ALWAQL$Q-NH2 1522.89 762.45 762.45 451 1187Ac-LAF$r8NYWANleL$Q-NH2 1570.89 786.45 786.5 452 1188Ac-LAF$r8NLWAQL$Q-NH2 1535.89 768.95 769.03 453 1189Ac-LAF$r8AYWANieL$A-NH2 1470.86 736.43 736.47 454 1190Ac-LAF$r8ALWAQL$A-NH2 1435.86 718.93 719.01 455 1191Ac-LAF$r8AYWAAL$A-NH2 1428.82 715.41 715.41 456 1192Ac-F$r8AYWEAc3cL$AAib-NH2 1399.75 700.88 700.95 457 1193Ac-F$r8AYWAQL$AA-NH2 1372.75 687.38 687.78 458 1194Ac-F$r8AYWAAc3cL$AA-NH2 1327.73 664.87 664.84 459 1195Ac-F$r8AYWSAc3cL$AA-NH2 1343.73 672.87 672.9 460 1196Ac-F$r8AYWEAc3cL$AS-NH2 1401.73 701.87 701.84 461 1197Ac-F$r8AYWEAc3cL$AT-NH2 1415.75 708.88 708.87 462 1198Ac-F$r8AYWEAc3cL$AL-NH2 1427.79 714.90 714.94 463 1199Ac-F$r8AYWEAc3cL$AQ-NH2 1442.76 722.38 722.41 464 1200Ac-F$r8AFWEAc3cL$AA-NH2 1369.74 685.87 685.93 465 1201Ac-F$r8AWWEAc3cL$AA-NH2 1408.75 705.38 705.39 466 1202Ac-F$r8AYWEAc3cL$SA-NH2 1401.73 701.87 701.99 467 1203Ac-F$r8AYWEAL$AA-NH2 1373.74 687.87 687.93 468 1204Ac-F$r8AYWENleL$AA-NH2 1415.79 708.90 708.94 469 1205Ac-F$r8AYWEAc3cL$AbuA-NH2 1399.75 700.88 700.95 470 1206Ac-F$r8AYWEAc3cL$NleA-NH2 1427.79 714.90 714.86 471 1207Ac-F$r8AYWEAibL$NleA-NH2 1429.80 715.90 715.97 472 1208Ac-F$r8AYWEAL$NleA-NH2 1415.79 708.90 708.94 473 1209Ac-F$r8AYWENeL$NleA-NH2 1457.83 729.92 729.96 474 1210Ac-F$r8AYWEAibL$AbU-NH2 1330.73 666.37 666.39 475 1211Ac-F$r8AYWENleL$AbU-NH2 1358.76 680.38 680.39 476 1212Ac-F$r8AYWEAL$AbU-NH2 1316.72 659.36 659.36 477 1213Ac-ITF$r8AFWAQL$S-NH2 1515.85 758.93 759.12 478 1214Ac-LTF$r8AWWAQL$S-NH2 1554.86 778.43 778.51 479 1215Ac-LTF$8AYWAQI$S-NH2 1531.84 766.92 766.96 480 1216Ac-LTF$r8AYWAQNle$S-NH2 1531.84 766.92 766.96 481 1217Ac-LTF$r8AYWAQLS$A-NH2 1602.88 802.44 802.48 482 1218Ac-LTF$r8AWWAQL$A-NH2 1538.87 770.44 770.89 483 1219Ac-LTF$8AYWAQI$A-NH2 1515.85 758.93 759.42 484 1220Ac-LTF$r8AYWAQNle$A-NH2 1515.85 758.93 759.42 485 1221Ac-LTF$r8AYWAQL$AA-NH2 1586.89 794.45 794.94 486 1222Ac-LTF$r8HWWAQL$S-NH2 1620.88 811.44 811.47 487 1223Ac-LTF$8HRWAQL$S-NH2 1590.90 796.45 796.52 488 1224 Ac-LTF$8HKWAQL$S-NH21562.90 782.45 782.53 489 1225 Ac-LTF$r8HYWAQLSW-NH2 1696.91 849.46849.5 491 1226 Ac-F$r8AYWAbUAL$A-NH2 1258.71 630.36 630.5 492 1227Ac-F$r8AbuYWEAL$A-NH2 1316.72 659.36 659.51 493 1228Ac-NlePRF%r8NYWRLL%QN-NH2 1954.13 978.07 978.54 494 1229Ac-TSF%r8HYWAQL%S-NH2 1573.83 787.92 787.98 495 1230Ac-LTF%r8AYWAQL%S-NH2 1533.86 767.93 768 496 1231 Ac-HTF$r8IIYWAQL$S-NH21621.84 811.92 811.96 497 1232 Ac-LHF$r8HYWAQL$S-NH2 1633.88 817.94818.02 498 1233 Ac-LTF$r8HHWAQL$S-NH2 1571.86 786.93 786.94 499 1234Ac-LTF$r8HYWHQL$S-NH2 1663.89 832.95 832.38 500 1235Ac-LTF$r8HYWAHL$S-NH2 1606.87 804.44 804.48 501 1236Ac-LTF$r8HYWAQL$H-NH2 1647.89 824.95 824.98 502 1237Ac-LTF$r8HYWAQL$S-NHPr 1639.91 820.96 820.98 503 1238Ac-LTF$r8HYWAQL$S-NHsBu 1653.93 827.97 828.02 504 1239Ac-LTF$r8HYWAQL$S-NHiBu 1653.93 827.97 828.02 505 1240Ac-LTF$r8HYWAQL$S-NHBn 1687.91 844.96 844.44 506 1241Ac-LTF$r8HYWAQL$S-NHPe 1700.92 851.46 851.99 507 1242Ac-LTF$r8HYWAQL$S-NHChx 1679.94 840.97 841.04 508 1243Ac-ETF$r8AYWAQL$S-NH2 1547.80 774.90 774.96 509 1244Ac-STF$r8AYWAQL$S-NH2 1505.79 753.90 753.94 510 1245Ac-LEF$r8AYWAQL$S-NH2 1559.84 780.92 781.25 511 1246Ac-LSF$r8AYWAQL$S-NH2 1517.83 759.92 759.93 512 1247Ac-LTF$r8EYWAQL$S-NH2 1589.85 795.93 795.97 513 1248Ac-LTF$r8SYWAQL$S-NH2 1547.84 774.92 774.96 514 1249Ac-LTF$r8AYWEQL$S-NH2 1589.85 795.93 795.9 515 1250Ac-LTF$r8AYWAEL$S-NH2 1532.83 767.42 766.96 516 1251Ac-LTF$r8AYWASL$S-NH2 1490.82 746.41 746.46 517 1252Ac-LTF$8AYWAQL$E-NH2 1573.85 787.93 787.98 518 1253Ac-LTF2CN$r8HYWAQL$S-NH2 1622.86 812.43 812.47 519 1254Ac-LTF3Cl$r8HYWAQL$S-NH2 1631.83 816.92 816.99 520 1255Ac-LTDip$r8HYWAQL$S-NH2 1673.90 837.95 838.01 521 1256Ac-LTF$r8HYWAQTle$S-NH2 1597.87 799.94 800.04 522 1257Ac-F$r8AY6clWEAL$A-NH2 1336.66 669.33 1338.56 523 1258Ac-F$r8AYdl6brWEAL$A-NH2 1380.61 691.31 692.2 524 1259Ac-F$r8AYdI6fWEAL$A-NH2 1320.69 661.35 1321.61 525 1260Ac-F$r8AYdl4mWEAL$A-NH2 1316.72 659.36 659.36 526 1261Ac-F$r8AYdl5clWEAL$A-NH2 1336.66 669.33 669.35 527 1262Ac-F$r8AYdl7mWEAL$A-NH2 1316.72 659.36 659.36 528 1263Ac-LTF%r8HYWAQL%A-NH2 1583.89 792.95 793.01 529 1264Ac-LTF$r8HCouWAQL$S-NH2 1679.87 840.94 841.38 530 1265Ac-LTFEHCouWAQLTS-NH2 1617.75 809.88 809.96 531 1266Ac-LTA$r8HCouWAQL$S-NH2 1603.84 802.92 803.36 532 1267Ac-F$r8AYWEAL$AbUA-NH2 1387.75 694.88 694.88 533 1268Ac-F$r8AYWEAI$AA-NH2 1373.74 687.87 687.93 534 1269Ac-F$r8AYWEANle$AA-NH2 1373.74 687.87 687.93 535 1270Ac-F$r8AYWEAmlL$AA-NH2 1429.80 715.90 715.97 536 1271Ac-F$r8AYWQAL$AA-NH2 1372.75 687.38 687.48 537 1272 Ac-F$r8AYWAAL$AA-NH21315.73 658.87 658.92 538 1273 Ac-F$r8AYWAbUAL$AA-NH2 1329.75 665.88665.95 539 1274 Ac-F$r8AYWNleAL$AA-NH2 1357.78 679.89 679.94 540 1275Ac-F$r8AbuYWEAL$AA-NH2 1387.75 694.88 694.96 541 1276Ac-F$r8NleYWEAL$AA-NH2 1415.79 708.90 708.94 542 1277Ac-FSr8FYWEAL$AA-NH2 1449.77 725.89 725.97 543 1278Ac-LTF$r8HYWAQhL$S-NH2 1611.88 806.94 807 544 1279Ac-LTF$r8HYWAQAdm$S-NH2 1675.91 838.96 839.04 545 1280Ac-LTF$r8HYWAQIgl$S-NH2 1659.88 830.94 829.94 546 1281Ac-F$r8AYWAQL$AA-NH2 1372.75 687.38 687.48 547 1282Ac-LTF$r8ALWAQL$Q-NH2 1522.89 762.45 762.52 548 1283Ac-F$r8AYWEAL$AA-NH2 1373.74 687.87 687.93 549 1284Ac-F$r8AYWENleL$AA-NH2 1415.79 708.90 708.94 550 1285Ac-F$r8AYWEAibL$Abu-NH2 1330.73 666.37 666.39 551 1286Ac-F$r8AYWENeL$AbU-NH2 1358.76 680.38 680.38 552 1287Ac-F$r8AYWEAL$Abu-NH2 1316.72 659.36 659.36 553 1288Ac-F$r8AYWEAc3cL$AbuA-NH2 1399.75 700.88 700.95 554 1289Ac-F$r8AYWEAc3cL$NleA-NH2 1427.79 714.90 715.01 555 1290H-LTF$8AYWAQL$S-NH2 1489.83 745.92 745.95 556 1291mdPEG3-LTF$rAYWAQL$S-NH2 1679.92 840.96 840.97 557 1292mdPEG7-LTF$r8AYWAQL$S-NH2 1856.02 929.01 929.03 558 1293Ac-F$r8ApmpEt6clWEAL$A-NH2 1470.71 736.36 788.17 559 1294Ac-LTF3Cl$r8AYWAQL$S-NH2 1565.81 783.91 809.18 560 1295Ac-LTF3Cl$r8HYWAQL$A-NH2 1615.83 808.92 875.24 561 1296Ac-LTF3Cl$r8HYWWQL$S-NH2 1746.87 874.44 841.65 562 1297Ac-LTF3Cl$r8AYWWQL$S-NH2 1680.85 841.43 824.63 563 1298Ac-LTF$r8AYWWQL$S-NH2 1646.89 824.45 849.98 564 1299Ac-ITF$r8HYWWQL$A-NH2 1696.91 849.46 816.67 565 1300Ac-LTF$8AYWWQL$A-NH2 1630.89 816.45 776.15 566 1301Ac-ITF4F$r8AYWAQL$S-NH2 1549.83 775.92 776.15 567 1302Ac-LTF2F$r8AYWAQL$S-NH2 1549.83 775.92 776.15 568 1303Ac-LTF3F$r8AYWAQL$S-NH2 1549.83 775.92 785.12 569 1304Ac-LTF34F2$r8AYWAQL$S-NH2 1567.83 784.92 785.12 570 1305Ac-LTF35F2$r8AYWAQL$S-NH2 1567.83 784.92 1338.74 571 1306Ac-F3Cl$r8AYWEAL$A-NH2 1336.66 669.33 705.28 572 1307Ac-F3Cl$r8AYWEAL$AA-NH2 1407.70 704.85 680.11 573 1308Ac-F$r8AY6clWEAL$AA-NH2 1407.70 704.85 736.83 574 1309Ac-F$r8AY6clWEAL$-NH2 1265.63 633.82 784.1 575 1310Ac-LTF$r8HYWAQL$t/S-NH2 16.03 9.02 826.98 576 1311Ac-LTF$r8HYWAQL$S-NHsBu 1653.93 827.97 828.02 577 1312Ac-STF$r8AYWAQL$S-NH2 1505.79 753.90 753.94 578 1313Ac-LTF$r8AYWAEL$S-NH2 1532.83 767.42 767.41 579 1314Ac-LTF$8AYWAQL$E-NH2 1573.85 787.93 787.98 580 1315mdPEG3-LTF$r8AYWAQL$S-NH2 1679.92 840.96 840.97 581 1316Ac-LTF$r8AYWAQhL$S-NH2 1545.86 773.93 774.31 583 1317Ac-LTF$r8AYWAQCha$S-NH2 1571.88 786.94 787.3 584 1318Ac-LTF$rAYWAQChg$S-NH2 1557.86 779.93 780.4 585 1319Ac-LTF$r8AYWAQCba$S-NH2 1543.84 772.92 780.13 586 1320Ac-LTF$r8AYWAQF$S-NH2 1565.83 783.92 784.2 587 1321Ac-LTF4F$r8HYWAQhL$S-NH2 1629.87 815.94 815.36 588 1322Ac-LTF4F$r8HYWAQCha$S-NH2 1655.89 828.95 828.39 589 1323Ac-ITFAF$r8HYWAQChg$S-NH2 1641.87 821.94 821.35 590 1324Ac-LTF4F$r8HYWAQCba$S-NH2 1627.86 814.93 814.32 591 1325Ac-LTF4F$r8AYWAQhL$S-NH2 1563.85 782.93 782.36 592 1326Ac-LTF4F$r8AYWAQCha$S-NH2 1589.87 795.94 795.38 593 1327Ac-LTF4F$r8AYWAQChg$S-NH2 1575.85 788.93 788.35 594 1328Ac-LTF4F$r8AYWAQCba$S-NH2 1561.83 781.92 781.39 595 1329Ac-LTF3Cl$r8AYWAQhL$S-NH2 1579.82 790.91 790.35 596 1330Ac-LTF3Cl$r8AYWAQCha$S-NH2 1605.84 803.92 803.67 597 1331Ac-LTF3Cl$r8AYWAQChg$S-NH2 1591.82 796.91 796.34 598 1332Ac-LTF3Cl$r8AYWAQCba$S-NH2 1577.81 789.91 789.39 599 1333Ac-LTF$8AYWAQhF$S-NH2 1579.84 790.92 791.14 600 1334Ac-LTF$r8AYWAQF3CF3$S-NH2 1633.82 817.91 818.15 601 1335Ac-LTF$r8AYWAQF3Me$S-NH2 1581.86 791.93 791.32 602 1336Ac-LTF$8AYWAQINal$S-NH2 1615.84 808.92 809.18 603 1337Ac-LTF$r8AYWAQBip$S-NH2 1641.86 821.93 822.13 604 1338Ac-LTF$r8FYWAQL$A-NH2 1591.88 796.94 797.33 605 1339Ac-LTF$r8HYWAQL$S-NHAm 1667.94 834.97 835.92 606 1340Ac-LTF$r8HYWAQL$S-NHiAm 1667.94 834.97 835.55 607 1341Ac-LTF$r8HYWAQL$S-NHnPr3Ph 1715.94 858.97 859.79 608 1342Ac-LTF$r8HYWAQL$S-NHnBu3,3Me 1681.96 841.98 842.49 610 1343Ac-LTF$r8HYWAQL$S-NHnPr 1639.91 830.96 821.58 611 1344Ac-LTF$r8HYWAQL$S-NHnEt2Ch 1707.98 854.99 855.35 612 1345Ac-LTF$r8HYWAQL$S-NHHex 1681.96 841.98 842.4 613 1346Ac-LTF$r8AYWAQL$S-NHmdPeg2 1633.91 817.96 818.35 614 1347Ac-LTF$8AYWAQL$A-NHmdPeg2 1617.92 809.96 810.3 615 1348Ac-LTF$r8AYWAQL$A-NHmdPeg4 1705.97 853.99 854.33 616 1349Ac-F$r8AYdI4mWEAL$A-NH2 1316.72 659.36 659.44 617 1350Ac-F$r8AYdlScIWEAL$A-NH2 1336.66 669.33 669.43 618 1351Ac-LThF$rAYWAQI$S-NH2 1545.86 773.93 774.11 619 1352Ac-LT2Nal$rAYWAQL$S-NH2 1581.86 791.93 792.43 620 1353Ac-LTA$r8AYWAQL$S-NH2 1455.81 728.91 729.15 621 1354Ac-LTF$rAYWVQL$S-NH2 1559.88 780.94 781.24 622 1355Ac-LTF$r8HYWAAL$A-NH2 1524.85 763.43 763.86 623 1356Ac-LTF$r8VYWAQL$A-NH2 1543.88 772.94 773.37 624 1357Ac-LTF$r8IYWAQL$S-NH2 1573.89 787.95 788.17 625 1358Ac-FTFSrVYWSQL$S-NH2 1609.85 805.93 806.22 626 1359Ac-ITF$r8FYWAQL$S-NH2 1607.88 804.94 805.2 627 1360Ac-2NalTF$r8VYWSQL$S-NH2 1659.87 830.94 831.2 628 1361Ac-ITF$r8LYWSQL$S-NH2 1589.89 795.95 796.13 629 1362Ac-FTF$r8FYWAQL$S-NH2 1641.86 821.93 822.13 630 1363Ac-WTF$r8VYWAQL$S-NH2 1632.87 817.44 817.69 631 1364Ac-WTF$r8WYWAQL$S-NH2 1719.88 860.94 861.36 632 1365Ac-VTF$r8AYWSQL$S-NH2 1533.82 767.91 768.19 633 1366Ac-WTF$r8FYWSQL$S-NH2 1696.87 849.44 849.7 634 1367Ac-FTF$r8IYWAQL$S-NH2 1607.88 804.94 805.2 635 1368Ac-WTF$r8VYWSQL$S-NH2 1648.87 825.44 824.8 636 1369 Ac-FTF$rLYWSQL$S-NH21623.87 812.94 812.8 637 1370 Ac-YTF$rFYWSQL$S-NH2 1673.85 837.93 837.8638 1371 Ac-LTF$r8AY6clWEAL$A-NH2 1550.79 776.40 776.14 639 1372Ac-LTF$r8AY6clWSQL$S-NH2 1581.80 791.90 791.68 640 1373Ac-F$r8AY6clWSAL$A-NH2 1294.65 648.33 647.67 641 1374Ac-F$r8AY6clWQAL$AA-NH2 1406.72 704.36 703.84 642 1375Ac-LHF$r8AYWAQL$S-NH2 1567.86 784.93 785.21 643 1376Ac-LTF$rAYWAQL$S-NH2 1531.84 766.92 767.17 644 1377 Ac-LTF$8AHWAQL$S-NH21505.84 753.92 754.13 645 1378 Ac-LTF$r8AYWAHL$S-NH2 1540.84 771.42771.61 646 1379 Ac-LTF$r8AYWAQLSH-NH2 1581.87 791.94 792.15 647 1380H-LTF$8AYWAQL$A-NH2 1473.84 737.92 737.29 648 1381 Ac-HHF$r8AYWAQL$S-NH21591.83 796.92 797.35 649 1382 Ac-aAibWTF$r8VYWSQL$S-NH2 1804.96 903.48903.64 650 1383 Ac-AibWTF$r8HYWAQL$S-NH2 1755.91 878.96 879.4 651 1384Ac-AibAWTF$r8HYWAQL$S-NH2 1826.95 914.48 914.7 652 1385Ac-fWTF$r8HYWAQL$S-NH2 1817.93 909.97 916.1 653 1386Ac-AibWWTF$r8HYWAQL$S-NH2 1941.99 972.00 972.2 654 1387Ac-WTF$r8LYWSQL$S-NH2 1662.88 832.44 832.8 655 1388Ac-WTF$r8NleYWSQL$S-NH2 1662.88 832.44 832.6 656 1389Ac-LTF$r8AYWSQL$a-NH2 1531.84 766.92 767.2 657 1390Ac-LTF$r8EYWARL$A-NH2 1601.90 801.95 802.1 658 1391 Ac-LTF$8EYWAHL$A-NH21582.86 792.43 792.6 659 1392 Ac-aTF$r8AYWAQL$S-NH2 1489.80 745.90746.08 660 1393 Ac-AibTF$r8AYWAQL$S-NH2 1503.81 752.91 753.11 661 1394Ac-AmfTF$r8AYWAQL$S-NH2 1579.84 790.92 791.14 662 1395Ac-AmwTF$r8AYWAQL$S-NH2 1618.86 810.43 810.66 663 1396Ac-NmLTF$r8AYWAQL$S-NH2 1545.86 773.93 774.11 664 1397Ac-LNmTF$r8AYWAQL$S-NH2 1545.86 773.93 774.11 665 1398Ac-LSarF$r8AYWAQL$S-NH2 1501.83 751.92 752.18 667 1399Ac-LGF$r8AYWAQL$S-NH2 1487.82 744.91 745.15 668 1400Ac-ITNmF$r8AYWAQL$S-NH2 1545.86 773.93 774.2 669 1401Ac-TF$rAYWAQL$S-NH2 1418.76 710.38 710.64 670 1402 Ac-ETF$r8AYWAQL$A-NH21531.81 766.91 767.2 671 1403 Ac-LTF$r8EYWAQL$A-NH2 1573.85 787.93 788.1672 1404 Ac-LT2Nal$r8AYWSQL$S-NH2 1597.85 799.93 800.4 673 1405Ac-LTF$rAYWAAL$S-NH2 1474.82 738.41 738.68 674 1406Ac-LTF$rAYWAQhCha$S-NH2 1585.89 793.95 794.19 675 1407Ac-LTF$r8AYWAQChg$S-NH2 1557.86 779.93 780.97 676 1408Ac-LTF$r8AYWAQCba$S-NH2 1543.84 772.92 773.19 677 1409Ac-LTF$r8AYWAQF3CF3$S-NH2 1633.82 817.91 818.15 678 1410Ac-LTF$r8AYWAQINal$S-NH2 1615.84 808.92 809.18 679 1411Ac-LTF$r8AYWAQBip$S-NH2 1641.86 821.93 822.32 680 1412Ac-LT2Nal$r8AYWAQL$S-NH2 1581.86 791.93 792.15 681 1413Ac-LTF$8AYWVQL$S-NH2 1559.88 780.94 781.62 682 1414Ac-LTF$r8AWWAQL$S-NH2 1554.86 778.43 778.65 683 1415Ac-FTF$r8VYWSQL$S-NH2 1609.85 805.93 806.12 684 1416Ac-ITF$r8FYWAQL$S-NH2 1607.88 804.94 805.2 685 1417Ac-ITF$r8LYWSQL$S-NH2 1589.89 795.95 796.22 686 1418Ac-FTF$r8FYWAQL$S-NH2 1641.86 821.93 822.41 687 1419Ac-VTF$r8AYWSQL$S-NH2 1533.82 767.91 768.19 688 1420Ac-LTF$rAHWAQL$S-NH2 1505.84 753.92 754.31 689 1421 Ac-LTF$8AYWAQL$H-NH21581.87 791.94 791.94 690 1422 Ac-LTF$r8AYWAHL$S-NH2 1540.84 771.42771.61 691 1423 Ac-aAibWTF$r8VYWSQL$S-NH2 1804.96 903.48 903.9 692 1424Ac-AibWTF$r8HYWAQL$S-NH2 1755.91 878.96 879.5 693 1425Ac-AibAWTF$r8HYWAQL$S-NH2 1826.95 914.48 914.7 694 1426Ac-fWTF$r8HYWAQL$S-NH2 1817.93 909.97 910.2 695 1427Ac-AibWWTF$r8HYWAQL$S-NH2 1941.99 972.00 972.7 696 1428Ac-WTF$r8LYWSQL$S-NH2 1662.88 832.44 832.7 697 1429Ac-WTF$r8NleYWSQL$S-NH2 1662.88 832.44 832.7 698 1430Ac-LTF$r8AYWSQL$a-NH2 1531.84 766.92 767.2 699 1431Ac-LTF$r8EYWARL$A-NH2 1601.90 801.95 802.2 700 1432 Ac-LTF$EYWAHL$A-NH21582.86 792.43 792.6 701 1433 Ac-aTF$rAYWAQL$S-NH2 1489.80 745.90 746.1702 1434 Ac-AibTF$r8AYWAQL$S-NH2 1503.81 752.91 753.2 703 1435Ac-AmfTF$F8AYWAQL$S-NH2 1579.84 790.92 791.2 704 1436Ac-AmwTF$r8AYWAQL$S-NH2 1618.86 810.43 810.7 705 1437Ac-NmLTF$r8AYWAQL$S-NH2 1545.86 773.93 774.1 706 1438Ac-LNmTF$r8AYWAQL$S-NH2 1545.86 773.93 774.4 707 1439Ac-LSarF$r8AYWAQL$S-NH2 1501.83 751.92 752.1 708 1440Ac-TF$r8AYWAQL$S-NH2 1418.76 710.38 710.8 709 1441 Ac-ETF$r8AYWAQL$A-NH21531.81 766.91 767.4 710 1442 Ac-LTF$r8EYWAQL$A-NH2 1573.85 787.93 788.2711 1443 Ac-WTF$r8VYWSQL$S-NH2 1648.87 825.44 825.2 /13 1444Ac-YTF$r8FYWSQL$S-NH2 1673.85 837.93 837.3 714 1445Ac-F$r8AY6dWSAL$A-NH2 1294.65 648.33 647.74 715 1446Ac-ETF$r8EYWVQL$S-NH2 1633.84 817.92 817.36 716 1447Ac-ETF$r8EHWAQL$A-NH2 1563.81 782.91 782.36 717 1448Ac-ITF$r8EYWAQL$S-NH2 1589.85 795.93 795.38 718 1449Ac-ITF$r8EHWVQL$A-NH2 1575.88 788.94 788.42 719 1450Ac-IT$r8EHWAQL$S-NH2 1563.85 782.93 782.43 720 1451Ac-LTF4F$r8AYWAQCba$S-NH2 1561.83 781.92 781.32 721 1452Ac-LTF3Cl$r8AYWAQhL$S-NH2 1579.82 790.91 790.64 722 1453Ac-LTF3Cl$r8AYWAQCha$S-NH2 1605.84 803.92 803.37 723 1454Ac-LTF3Cl$r8AYWAQChg$S-NH2 1591.82 796.91 796.27 724 1455Ac-LTF3Cl$r8AYWAQCba$S-NH2 1577.81 789.91 789.83 725 1456Ac-LTF$r8AY6clWSQL$S-NH2 1581.80 791.90 791.75 726 1457Ac-LTF4F$r8HYWAQhL$S-NH2 1629.87 815.94 815.36 727 1458Ac-LTF4F$r8HYWAQCba$S-NH2 1627.86 814.93 814.32 728 1459Ac-LTF4F$r8AYWAQhL$S-NH2 1563.85 782.93 782.36 729 1460Ac-LTF4F$r8AYWAQChg$S-NH2 1575.85 788.93 788.35 730 1461Ac-ETF$r8EYWVAL$S-NH2 1576.82 789.41 788.79 731 1462Ac-ETF$r8EHWAAL$A-NH2 1506.79 754.40 754.8 732 1463Ac-ITF$r8EYWAAL$S-NH2 1532.83 767.42 767.75 733 1464Ac-ITi$r8EHWVAI$A-NH2 1518.86 760.43 760.81 734 1465Ac-ITFSrEHWAAL$S-NH2 1506.82 754.41 754.8 735 1466Pam-ITF$r8EYWAQL$S-NH2 1786.07 894.04 894.48 736 1467Pam-ETF$r8EYWAQL$S-NH2 1802.03 902.02 902.34 737 1468Ac-LTF$8AYWLQL$S-NH2 1573.89 787.95 787.39 738 1469Ac-LTF$r8EYWLQL$S-NH2 1631.90 816.95 817.33 739 1470Ac-LTF$r8EHWLQL$S-NH2 1605.89 803.95 804.29 740 1471Ac-LTF$r8VYWAQL$S-NH2 1559.88 780.94 781.34 741 1472Ac-LTF$8AYWSQL$S-NH2 1547.84 774.92 775.33 742 1473Ac-ETF$r8AYWAQL$S-NH2 1547.80 774.90 775.7 743 1474Ac-LTF$r8EYWAQL$S-NH2 1589.85 795.93 796.33 744 1475Ac-LTF$r8HYWAQL$S-NHAm 1667.94 834.97 835.37 745 1476Ac-LTF$r8HYWAQL$S-NHiAm 1667.94 834.97 835.27 746 1477Ac-LTF$r8HYWAQL$S-NHnPr3Ph 1715.94 858.97 859.42 747 1478Ac-LTF$r8HYWAQL$S-NHnBu3,3Me 1681.96 841.98 842.67 748 1479Ac-LTF$r8HYWAQL$S-NHnBu 1653.93 827.97 828.24 749 1480Ac-LTF$r8HYWAQL$S-NHnPr 1639.91 820.96 821.31 750 1481Ac-LTF$r8HYWAQL$S-NHnEt2Ch 1707.98 854.99 855.35 751 1482Ac-LTF$r8HYWAQL$S-NHHex 1681.96 841.98 842.4 752 1483Ac-LTF$8AYWAQL$S-NHmdPeg2 1633.91 817.96 855.35 753 1484Ac-LTF$r8AYWAQL$A-NHmdPeg2 1617.92 809.96 810.58 754 1485Ac-LTF$r5AYWAAL$s8S-NH2 1474.82 738.41 738.79 755 1486Ac-LTF$r8AYWCOuQL$S-NH2 1705.88 853.94 854.61 756 1487Ac-LTF$r8CouYWAQL$S-NH2 1705.88 853.94 854.7 757 1488Ac-CouTF$r8AYWAQL$S-NH2 1663.83 832.92 833.33 758 1489H-LTF$r8AYWAQL$A-NH2 1473.84 737.92 737.29 759 1490Ac-HHF$r8AYWAQL$S-NH2 1591.83 796.92 797.72 760 1491Ac-LT2Nal$r8AYWSQL$S-NH2 1597.85 799.93 800.68 761 1492Ac-LTF$r8HCouWAQL$S-NH2 1679.87 840.94 841.38 762 1493Ac-LTF$r8AYWCOu2QL$S-NH2 1789.94 895.97 896.51 763 1494Ac-LTF$r8COu2YWAQL$S-NH2 1789.94 895.97 896.5 764 1495Ac-Cou2TF$r8AYWAQL$S-NH2 1747.90 874.95 875.42 765 1496Ac-LTF$r8ACou2WAQL$S-NH2 1697.92 849.96 850.82 766 1497Dmaac-LTF$r8AYWAQL$S-NH2 1574.89 788.45 788.82 767 1498Hexac-LTF$rAYWAQL$S-NH2 1587.91 794.96 795.11 768 1499Napac-ITF$r8AYWAQL$S-NH2 1657.89 829.95 830.36 769 1500Pam-LTF$rAYWAQL$S-NH2 1728.06 865.03 865.45 770 1501Ac-LT2NalSrHYAAQL$S-NH2 1532.84 767.42 767.61 771 1502Ac-LT2Nal$/r8HYWAQL$/S-NH2 1675.91 838.96 839.1 772 1503Ac-LT2Nal$r8HYFAQL$S-NH2 1608.87 805.44 805.9 773 1504Ac-LT2Nal$r8HWAAQL$S-NH2 1555.86 778.93 779.08 774 1505Ac-LT2NalSrHYAWQL$S-NH2 1647.88 824.94 825.04 775 1506Ac-LT2Nal$r8HYAAQW$S-NH2 1605.83 803.92 804.05 776 1507Ac-LTW$r8HYWAQL$S-NH2 1636.88 819.44 819.95 777 1508Ac-LT1Nal$r8HYWAQL$S-NH2 1647.88 824.94 825.41

In some embodiments, the peptidomimetic macrocycles disclosed herein donot comprise a peptidomimetic macrocycle structure as shown in Table 2b.

Table 2c shows examples of non-crosslinked polypeptides comprisingD-amino acids.

TABLE 2c SEQ ID Exact Found Calc Cak Cak SP NO: Sequence Isomer MassMass (M + 1)/1 (M + 2)/2 (M + 3)/3 SP778 1509 Ac-tawyanfekllr-NH2 777.46SP779 1510 Ac-tawyanf4CF3ekllr- 811.41 NH2

Peptidomimetic macrocycles can also be prepared that target or interactwith proteins that a virus needs for infection or replication within ahost cell. Such viruses can be, for example, influenza viruses belongingto Orthomyxoviridae family of viruses. This family also includes Thogotoviruses and Dhoriviruses. There are several types and subtypes ofinfluenza viruses known, which infect humans and other species.Influenza type A viruses infect people, birds, pigs, horses, seals andother animals, but wild birds are the natural hosts for these viruses.Influenza type A viruses are divided into subtypes and named on thebasis of two proteins on the surface of the virus: hemagglutinin (HA)and neuraminidase (NA). For example, an “H7N2 virus” designates aninfluenza A subtype that has an HA7 protein and an NA2 protein.Similarly an “H5N1” virus has an HA 5 protein and an NA1 protein. Thereare 16 known HA subtypes and 9 known NA subtypes. Many differentcombinations of HA and NA proteins are possible. Only some influenza Asubtypes (i.e., H1N1, H1N2, and H3N2) are currently in generalcirculation among people. Other subtypes are found most commonly inother animal species. For example, H7N7 and H3N8 viruses cause illnessin horses, and H3N8 also has recently been shown to cause illness indogs.

Antiviral agents according to the invention can be used to protecthigh-risk groups (hospital units, institutes caring for elderly,immuno-suppressed individuals), and on a case by case basis. A potentialuse for antiviral agents is to limit the spread and severity of thefuture pandemics whether caused by avian H5N1 or other strains ofinfluenza virus. Avian influenza A viruses of the subtypes H5 and H7,including H5N1, H7N7, and H7N3 viruses, have been associated with highpathogenicity, and human infection with these viruses have ranged frommild (H7N3, H7N7) to severe and fatal disease (H7N7, H5N1). Humanillness due to infection with low pathogenicity viruses has beendocumented, including very mild symptoms (e.g., conjunctivitis) toinfluenza-like illness. Examples of low pathogenicity viruses that haveinfected humans include H7N7, H9N2, and H7N2.

Influenza B viruses are usually found in humans but can also infectseals. Unlike influenza A viruses, these viruses are not classifiedaccording to subtype. Influenza B viruses can cause morbidity andmortality among humans, but in general are associated with less severeepidemics than influenza A viruses. Although influenza type B virusescan cause human epidemics, they have not caused pandemics.

Influenza type C viruses cause mild illness in humans and do not causeepidemics or pandemics. These viruses can also infect dogs and pigs.These viruses are not classified according to subtype.

Influenza viruses differ from each other in respect to cell surfacereceptor specificity and cell tropism, however they use common entrypathways. Charting these pathways and identification of host cellproteins involved in virus influenza transmission, entry, replication,biosynthesis, assembly, or exit allows the development of general agentsagainst existing and emerging strains of influenza. The agents can alsoprove useful against unrelated viruses that use similar pathways. Forexample, the agents can protect airway epithelial cells against a numberof different viruses in addition to influenza viruses.

In one embodiment the targeted virus is an adenovirus. Adenoviruses mostcommonly cause respiratory illness; symptoms of respiratory illnesscaused by adenovirus infection range from the common cold syndrome topneumonia, croup, and bronchitis. Patients with compromised immunesystems are especially susceptible to severe complications of adenovirusinfection. Acute respiratory disease (ARD), first recognized amongmilitary recruits during World War II, can be caused by adenovirusinfections during conditions of crowding and stress. Adenoviruses aremedium-sized (90-100 nm), nonenveloped icosohedral viruses containingdouble-stranded DNA. There are 49 immunologically distinct types (6subgenera: A through F) that can cause human infections. Adenovirusesare unusually stable to chemical or physical agents and adverse pHconditions, allowing for prolonged survival outside of the body. Someadenoviruses, such as AD2 and Ad5 (species C) use clathrin mediatedendocytosis and macropinocytosis for infectious entry. Otheradenoviruses, such as Ad3 (species B) use dynamin dependent endocytosisand macropinocytosis for infectious entry.

In one embodiment the targeted virus is a respiratory syncytial virus(RSV). RSV is the most common cause of bronchiolitis and pneumonia amonginfants and children under 1 year of age. Illness begins most frequentlywith fever, runny nose, cough, and sometimes wheezing. During theirfirst RSV infection, between 25% and 40% of infants and young childrenhave signs or symptoms of bronchiolitis or pneumonia, and 0.5% to 2%require hospitalization. Most children recover from illness in 8 to 15days. The majority of children hospitalized for RSV infection are under6 months of age. RSV also causes repeated infections throughout life,usually associated with moderate-to-severe cold-like symptoms: however,severe lower respiratory tract disease can occur at any age, especiallyamong the elderly or among those with compromised cardiac, pulmonary, orimmune systems. RSV is a negative-sense, enveloped RNA virus. The virionis variable in shape and size (average diameter of between 120 and 300nm), is unstable in the environment (surviving only a few hours onenvironmental surfaces), and is readily inactivated with soap and waterand disinfectants.

In one embodiment the targeted virus is a human parainfluenza virus(HPIV). HPIVs are second to respiratory syncytial virus (RSV) as acommon cause of lower respiratory tract disease in young children.Similar to RSV, HPIVs can cause repeated infections throughout life,usually manifested by an upper respiratory tract illness (e.g., a coldand/or sore throat). HPIVs can also cause serious lower respiratorytract disease with repeat infection (e.g., pneumonia, bronchitis, andbronchiolitis), especially among the elderly, and among patients withcompromised immune systems. Each of the four HPIVs has differentclinical and epidemiologic features. The most distinctive clinicalfeature of HPIV-1 and HPIV-2 is croup (i.e., laryngotracheobronchitis):HPIV-1 is the leading cause of croup in children, whereas HPIV-2 is lessfrequently detected. Both HPIV-1 and -2 can cause other upper and lowerrespiratory tract illnesses. HPIV-3 is more often associated withbronchiolitis and pneumonia. HPIV-4 is infrequently detected, possiblybecause it is less likely to cause severe disease. The incubation periodfor HPIVs is generally from 1 to 7 days. HPIVs are negative-sense,single-stranded RNA viruses that possess fusion andhemagglutinin-neuraminidase glycoprotein “spikes” on their surface.There are four serotypes types of HPIV (1 through 4) and two subtypes(4a and 4b). The virion varies in size (average diameter between 150 and300 nm) and shape, is unstable in the environment (surviving a few hourson environmental surfaces), and is readily inactivated with soap andwater.

In one embodiment the targeted virus is a coronavirus. Coronavirus is agenus of animal virus belonging to the family Coronaviridae.Coronaviruses are enveloped viruses with a positive-sensesingle-stranded RNA genome and a helical symmetry. The genomic size ofcoronaviruses ranges from approximately 16 to 31 kilobases,extraordinarily large for an RNA virus. The name “coronavirus” isderived from the Latin corona, meaning crown, as the virus envelopeappears under electron microscopy to be crowned by a characteristic ringof small bulbous structures. This morphology is actually formed by theviral spike peplomers, which are proteins that populate the surface ofthe virus and determine host tropism. Coronaviruses are grouped in theorder Nidovirales, named for the Latin nidus, meaning nest, as allviruses in this order produce a 3′ co-terminal nested set of subgenomicmRNAs during infection. Proteins that contribute to the overallstructure of all coronaviruses are the spike, envelope, membrane andnucleocapsid. In the specific case of SARS a defined receptor-bindingdomain on S mediates the attachment of the virus to its cellularreceptor, angiotensin-converting enzyme 2.

In one embodiment the targeted virus is a rhinovirus. Rhinovirus is agenus of the Picomaviridae family of viruses. Rhinoviruses are the mostcommon viral infective agents in humans, and a causative agent of thecommon cold. There are over 105 serologic virus types that cause coldsymptoms, and rhinoviruses are responsible for approximately 50% of allcases. Rhinoviruses have single-stranded positive sense RNA genomes ofbetween 7.2 and 8.5 kb in length. At the 5′ end of the genome is avirus-encoded protein, and like mammalian mRNA, there is a 3′ poly-Atail. Structural proteins are encoded in the 5′ region of the genome andnonstructural at the end. This is the same for all picornaviruses. Theviral particles themselves are not enveloped and are icosahedral instructure.

Any secondary structure of a viral protein (or of a host cell proteininvolved in viral infectivity) can form the basis of the methods. Forexample, a viral protein comprising a secondary structure which is ahelix can be used to design peptidomimetic macrocycles based on thehelix.

In one embodiment, the peptidomimetic macrocycle is designed based onthe PB1 or PB2 sequence of an influenza virus. The PB1 sequence ishighly conserved across all known strains of influenza A virus, whichcan result in less drug resistance should than that observed with thecurrent standard of care. An alignment of the first 25 N-terminal aminoacids of PB1 from the NCBI data bank's 2,485 influenza A virus strains(Ghanem, 2007) demonstrates the remarkable sequence conservation in thePA interaction domain of PB1. Therefore, antiviral therapies based onthe PB1 sequence can block most, if not all, influenza A virus strains.Additionally, sequence modification of a peptidomimetic macrocycle basedon these few variations in PB1 can enable an antiviral cocktail of PB1inhibitors to eliminate resistance due to escape mutants.

Table 3a shows a list of peptidomimetic macrocycles derived from thePA-binding helix of PB1 that were prepared.

Table 3b shows a list of selected peptidomimetic macrocycles from Table3a. SP-791 and SP-794 were prepared by increasing the length and alaninecontent (%) of the SP-786 sequence. These modifications led to afive-fold increase in antiviral activity compared to that of SP-786.SP-798 was prepared by incorporating an i, i+7 crosslink instead of thei, i+4 crosslink of SP-786. SP-192 exhibited improved anti-viralactivity (EC₅₀=4.5 mM) compared to that of SP-786.

In some embodiments, the invention provides a peptidomimetic macrocyclethat comprises an amino acid sequence that has at least 60%, 70%, 80%,90%, 95%, 97%, or 100% identity to any one of the amino acid sequencesin Table 3a or 3b.

TABLE 3a Prepared peptidomimetic macrocycles derived from the PA-bindinghelix of pB1. SEQ ID SP# NO: 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17780 1511 Ac— Nle D V N $ T L L $ L K V Aib A Q —NH₂ 781 1512 Ac— Nle D VN $ T L L $ L K A Aib A Q —NH₂ 782 1513 Ac— Nle D V N $ T L L $ L K VAib A A —NH₂ 783 1514 Ac— Nle D V N $ T L L $ L K V P Aib Q —NH₂ 7841515 Ac— F D V N $ T L L $ L K V Aib A Q —NH₂ 785 1516 5-FAM— Ba Nle D VN $ T L L $ L A V Aib A Q —NH₂ 786 1517 Ac— Nle D V N $ T L L $ L A VAib A Q —NH₂ 787 1518 Ac— Nle D V N $ T L L $ L A V Aib A Q A A —NH₂ 7881519 Ac— Nle D V N $ T L L $ L A V A A Q A A —NH₂ 789 1520 Ac— Nle D V N$ T L L $ L A V A A A A A —NH₂ 790 1521 Ac— Nle D V N $ T L L $ L A V SA Q A A —NH₂ 791 1522 Ac— Nle D V N $ T L L $ L A V Q A Q A A —NH₂ 7921523 Ac— Nle D V N $ T L L $ L A V E A Q A A —NH₂ 793 1524 Ac— Nle D V N$ T L L $ L A V S A A A A —NH₂ 794 1525 Ac— Nle D V N $ T L L $ L A V QA A A A —NH₂ 795 1526 Ac— Nle D V N $ T L L $ L A V E A A A A —NH₂ 7961527 Ac— Nle D V N $ T L L $ L A V Aib A Q A a —NH₂ 797 1528 Ac— Nle D VN $ T L L $ L A V Aib A Q A a —OH 798 1529 Ac— Nle D V N $r8 T L L F L A$ A A Q —NH₂ 799 1530 Ac— Nle D V N $ T L L $ L A V Q Aib Q A A —NH₂ 8001531 Ac— Nle D V N $ T L L $ L A V Q A Q Aib A —NH₂ 801 1532 Ac— Nle D VN $ T L L $ L A V Q Aib A A A —NH₂ 802 1533 Ac— Nle D V N $ T L L $ L AV Q A A Aib A —NH₂ 803 1534 Ac— Nle D V N $ T L L $ L A V Q A Aib A A—NH₂ 804 1535 Ac— Nle D V N $r8 T L L F L A $ A A Q A A —NH₂ 805 1536Ac— Nle D V N $r8 T L L F L A $ A A A A A —NH₂ 806 1537 Ac— Nle D V N$r8 T L L F L A $ Q A Q A A —NH₂ 807 1538 Ac— Nle D V N $r8 T L L F L A$ Q A A A A —NH₂

TABLE 3b Selected peptidomimetic macrocycles derived from the PA-bindinghelix of pB1. RT Ala CPE, EC₅₀ SP# Crosslink Ch L VH (min)* (%) (μM)**786 i, i + 4 −1 15 4.7 10.68 13 23 791 i, i + 4 −1 17 7.2 8.02 24 4.1794 i, i + 4 −1 17 5.1 9.54 29 6.1 799 i, i + 4 −1 17 7 18 <6.1*** 800i, i + 4 −1 17 7 8.47 18 <6.1*** 801 i, i + 4 −1 17 4.9 10.01 24 <6.1***802 i, i + 4 −1 17 4.9 10.18 24 <6.1*** 803 i, i + 4 −1 17 4.9 10.26 24<6.1*** 798 i, i + 7 −1 15 4.9 10.18 20 4.5 804 i, i + 7 −1 17 5.1 10.9229 <4.5*** 805 i, i + 7 −1 17 3 13.03 35 <4.5*** 806 i, i + 7 −1 17 7.28.86 24 <4.5*** 807 i, i + 7 −1 17 5.1 10.83 29 <4.5*** *See Example 11table **By Neutral Red assay (Influenza A Virus H1N1 California/07/2009)***Predicted Ch = net charge; L = length in amino acids; VH = vonHeijne; RT = retention time; Ala = alanine content

In some embodiments of the invention, the peptide sequence is derivedfrom the BCL-2 family of proteins. The BCL-2 family is defined by thepresence of up to four conserved BCL-2 homology (BH) domains designatedBH1, BH2, BH3, and BH4, all of which include α-helical segments(Chittenden et al. (1995), EMBO 14:5589; Wang et al. (1996), Genes Dev.10:2859). Anti-apoptotic proteins, such as BCL-2 and BCL-X_(L), displaysequence conservation in all BH domains. Pro-apoptotic proteins aredivided into “multidomain” family members (e.g., BAK, BAX), whichpossess homology in the BH1, BH2, and BH3 domains, and “BH3-domain only”family members (e.g., BID, BAD, BIM, BIK, NOXA, PUMA), that containsequence homology exclusively in the BH3 amphipathic α-helical segment.BCL-2 family members have the capacity to form homo- and heterodimers,suggesting that competitive binding and the ratio between pro- andanti-apoptotic protein levels dictates susceptibility to death stimuli.Anti-apoptotic proteins function to protect cells from pro-apoptoticexcess, i.e., excessive programmed cell death. Additional “security”measures include regulating transcription of pro-apoptotic proteins andmaintaining them as inactive conformers, requiring either proteolyticactivation, dephosphorylation, or ligand-induced conformational changeto activate pro-death functions. In certain cell types, death signalsreceived at the plasma membrane trigger apoptosis via a mitochondrialpathway. The mitochondria can serve as a gatekeeper of cell death bysequestering cytochrome c, a critical component of a cytosolic complexwhich activates caspase 9, leading to fatal downstream proteolyticevents. Multidomain proteins such as BCL-2/BCL-X_(L) and BAK/BAX playdueling roles of guardian and executioner at the mitochondrial membrane,with their activities further regulated by upstream BH3-only members ofthe BCL-2 family. For example, BID is a member of the BH3-domain onlyfamily of pro-apoptotic proteins, and transmits death signals receivedat the plasma membrane to effector pro-apoptotic proteins at themitochondrial membrane. BID has the capability of interacting with bothpro- and anti-apoptotic proteins, and upon activation by caspase 8,triggers cytochrome c release and mitochondrial apoptosis. Deletion andmutagenesis studies determined that the amphipathic α-helical BH3segment of pro-apoptotic family members can function as a death domainand thus can represent a critical structural motif for interacting withmultidomain apoptotic proteins. Structural studies have shown that theBH3 helix can interact with anti-apoptotic proteins by inserting into ahydrophobic groove formed by the interface of BH1, 2 and 3 domains.Activated BID can be bound and sequestered by anti-apoptotic proteins(e.g., BCL-2 and BCL-X_(L)) and can trigger activation of thepro-apoptotic proteins BAX and BAK, leading to cytochrome c release anda mitochondrial apoptosis program. BAD is also a BH3-domain onlypro-apoptotic family member whose expression triggers the activation ofBAX/BAK. In contrast to BID, however, BAD displays preferential bindingto anti-apoptotic family members, BCL-2 and BCL-X_(L). Whereas the BADBH3 domain exhibits high affinity binding to BCL-2, BAD BH3 peptide isunable to activate cytochrome c release from mitochondria in vitro,suggesting that BAD is not a direct activator of BAX/BAK. Mitochondriathat over-express BCL-2 are resistant to BID-induced cytochrome crelease, but co-treatment with BAD can restore BID sensitivity.Induction of mitochondrial apoptosis by BAD appears to result fromeither: (1) displacement of BAX/BAK activators, such as BID and BID-likeproteins, from the BCL-2/BCL-X_(L) binding pocket, or (2) selectiveoccupation of the BCL-2/BCL-X_(L) binding pocket by BAD to preventsequestration of BID-like proteins by anti-apoptotic proteins. Thus, twoclasses of BH3-domain only proteins have emerged, BID-like proteins thatdirectly activate mitochondrial apoptosis, and BAD-like proteins, thathave the capacity to sensitize mitochondria to BID-like pro-apoptoticsby occupying the binding pockets of multidomain anti-apoptotic proteins.Various α-helical domains of BCL-2 family member proteins amenable tothe methodology disclosed herein have been disclosed (Walensky et al.(2004), Science 305:1466: and Walensky et al., U.S. Patent PublicationNo. 2005/0250680, the entire disclosures of which are incorporatedherein by reference).

Myeloid cell leukemia 1 (MCL-1) is a protein that inhibits cell deaththrough the binding and inhibition of pro-death factors such as BCL-2interacting mediator (BIM). When MCL-1 is over-expressed, the rate ofcell death in a cell or tissue is reduced. In some embodiments, thepeptide sequences are derived from BIM. In some embodiments, apeptidomimetic macrocycle peptide derived from a human BIM peptide canbe a peptide comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, or 22 amino acids from a BIM peptidesequence.

In some embodiments, a peptidomimetic macrocycle peptide derived from ahuman BIM peptide sequence can be a peptide comprising 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22 aminoacids that are different from the selected sequences from which thepeptide is derived. In some embodiments. a peptidomimetic macrocyclepeptide derived from a human BIM peptide sequence can be a peptidecomprising a mutation at amino acid position 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22. In someembodiments, mutations are mutations of non-essential amino acids. Insome embodiments, mutations are mutations of essential amino acids. Insome embodiments, mutations are mutations of hydrophobic amino acids. Insome embodiments, mutations are mutations of naturally occurring aminoacids. In some embodiments, mutations are mutations to a conservativeamino acid. In some embodiments, a peptidomimetic macrocycle peptidederived from a human BIM peptide sequence can be a peptide comprising 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,or 22 amino acid analogues. In some embodiments, a peptidomimeticmacrocycle peptide derived from a human BIM peptide sequence can be apeptide comprising 1 or 2 capping groups.

In some embodiments, the peptidomimetic macrocycle comprises aC-terminal truncation of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 aminoacids from an amino acid sequence of BIM In some embodiments, thepeptidomimetic macrocycle comprises a N-terminal truncation of 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22amino acids from the sequence of BIM.

A non-limiting list of suitable BIM macrocycles for use in the presentdisclosure are given in Tables 4a and 4b. In Tables 4a and 4b, at theC-terminus, some peptides possess a carboxamide terminus (shown as—NH₂): some peptides possess a hydroxyl terminus (shown as —OH); somepeptides possess a 5-carboxyfluorescein terminus (shown as -5-FAM); somepeptides possess a isobutylamide terminus (shown as —NHiBu); somepeptides possess a cyclohexylamide terminus (shown as —NHChx); somepeptides possess a cyclohexylmethylamide terminus (shown as —NHMeChx);some peptides possess a phenethylamide terminus (shown as —NHPe); somepeptides possess a n-butylamide terminus (shown as —NHBu); some peptidespossess a sec-butylamide terminus (shown as —NHsBu); and some peptidespossess an uncapped terminus (shown as no terminal modification).

In Tables 4a and 4b, at the N-terminus, some peptides possess an acetylterminus (shown as Ac-); some peptides possess a fluoresceinisothiocyanate terminus (shown as FITC-); some peptides possess asingle-unit polyethylene glycol terminus (shown as dPEG1-); somepeptides possess a five-unit polyethylene glycol terminus (shown asdPEG5-); some peptides possess an eleven-unit polyethylene glycolterminus (shown as dPEG11-); some peptides possess a propyl terminus(shown as Pr-); some peptides possess a biotin terminus (shown asBiotin-): some peptides possess a KLH terminus (shown as KLH-); somepeptides possess an ovalbumin terminus (shown as OVA-); some peptidespossess an uncapped terminus (shown as H-); some peptides possess aisobutyl terminus (shown as iBu-); some peptides possess a decanoylterminus (shown as Decac-); some peptides possess a benzyl terminus(shown as Bz-); some peptides possess a cyclohexyl terminus (shown asChx-); some peptides possess a benzyl terminus (shown as Bz-); somepeptides possess a Vrl terminus (shown as Vrl-); some peptides possess aHBS terminus (shown as HBS-): some peptides possess a Melm terminus(shown as MeImC-); some peptides possess a tert-butyl terminus (shown ast-Bu-U-); some peptides possess a nonanoyl terminus (shown as non-U-);some peptides possess a ethyl terminus (shown as Et-U-); some peptidespossess a cyclohexyl terminus (shown as Chx-U-): some peptides possess aisopropyl terminus (shown as iPr-U-): some peptides possess a phenylterminus (shown as Ph-U-): some peptides possess a uric terminus (shownas NH₂CO-); some peptides possess a palmitoyl terminus (shown as Pam-):some peptides possess a heptenoic terminus (shown as Hep-); and somepeptides possess a 5-carboxytetramethylrhodamine terminus (shown as5-TAMRA-).

Table 4a shows a list of peptidomimetic macrocycles derived from theMCL-1/BCL-X_(L)/BCL-2-binding helix of BIM that were prepared. Table 4bshows a list of selected peptidomimetic macrocycles from Table 4a.SP-809 was prepared by incorporating an i, i+7 crosslink into thesequence of the linear peptide LP-2. SP-815 was prepared by removal ofthe two terminal arginine residues and an alanine substitution atposition 13 of SP-809. SP-962 was prepared by a homoleucine substitutionat position 9 and a F4F at position 17 of SP-815.

In some embodiments, the invention provides a peptidomimetic macrocyclethat comprises an amino acid sequence that has at least 60%, 70%, 80%,90%, 95%, 97%, or 100% identity to any one of the amino acid sequencesin Table 4a or 4b.

TABLE 4a Prepared peptidomimetic macrocycles derived from the MCL-1/BCL-X1/BCL-2-binding helix of BIM SEQ ID SP# NO: 1 2 3 4 5 6 7 8 9 10 1112 13 14 15 LP2 1539 Ac— I W I A Q E L R R I G D E F N 808 1540 Ac— I WI A Q E L R $r8 I G D E F N 809 1541 Ac— I W I A Q A L R $r8 I G D E F N810 1542 Ac— I W I A Q E L R $r8 I G D E F N 812 1543 Ac— W I A Q A L R$r8 I G D A F N 813 1544 Ac— I A Q A L R $r8 I G D A F A 814 1545 Ac— IA Q A L R $r8 I G D A F N 815 1546 Ac— I W I A Q A L R $r8 I G D A F N816 1547 Ac— W I A Q A L R $r8 I G D A F N 817 1548 Ac— I A Q A L R $r8I G D A F N 818 1549 Ac— I A A A L R $r8 I G D A F N 819 1550 Ac— I A QA L A $r8 I G D A F N 820 1551 Ac— I A Q A L R $r8 I A D A F N 821 1552Ac— I A Q A L R $r8 I G D A A N 822 1553 Ac— I A Q A L R $r8 I G D A F N823 1554 Ac— I $ I A Q $ L R $r8 I G D E F N 824 1555 Ac— I W I A Q A LR %r8 I G D A F N 825 1556 Ac— I W I A Q A L R $r8 I G D E F A 826 1557Ac— I W I A Q A L R $r8 I G D Q A N 827 1558 FITC— Ba I W I A Q A L R$r8 I G D A F N 828 1559 5-FAM— Ba I W I A Q A L R $r8 I G D A F N 8291560 5-FAM— Ba I W I A Q A L R $r8 I G D E F N 830 1561 Ac— I A I A Q AL R $r8 I G D A F N 831 1562 Ac— I W I A Q A L R $r8 I G D E F N 8321563 Ac— I W I A Q A L R $r8 I G D Q F N 833 1564 Ac— I W I A A A L R$r8 I G D E F N 834 1565 Ac— I W I A A A L R $r8 I G D Q F N 835 1566Ac— I W I A A A L R $r8 I G D A F N 836 1567 Ac— I W I A Q A L R $r8 I GD A F A 837 1568 Ac— I W I A Q A L Cit $r8 I G D A F N 838 1569 Ac— I WI A Q A L Cit $r8 I G D Q F N 839 1570 Ac— I W I A Q A L H $r8 I G D A FN 840 1571 Ac— I W I A Q A L H $r8 I G D Q F N 841 1572 Ac— I W I A Q AL Q $r8 I G D A F N 842 1573 Ac— I W I A Q A L Q $r8 I G D Q F N 8431574 Ac— I W I A Q A L R $r8 I G D A A N 844 1575 Ac— I W I A Q A L R$r8 I G D A I N 845 1576 Ac— I W I A Q A L R $r8 I G D Q I N 846 1577Ac— I W I A Q A A R $r8 I G D A A N 847 1578 Ac— I W I A Q A L R $r8 I AD A F N 848 1579 Ac— I W I A Q A L R $r8 I A D Q F N 849 1580 Ac— I W IA Q A L R $r8 A G D A F N 850 1581 Ac— I W I A Q A L R $r8 A G D Q F N851 1582 Ac— I W I A Q A L R $r8 I G D A F N 852 1583 Ac— I W I A Q A LR $r8 I G D Q F N 853 1584 Ac— I W F A Q A L R $r8 I G D A F N 854 1585Ac— I W F A Q A L R $r8 I G D Q F N 855 1586 Ac— I W I A Q A L A $r8 I GD A F N 856 1587 Ac— I W I A Q A L R $r8 I G N A F N 857 1588 Ac— I W IA Q A A R $r8 I G D A F N 858 1589 Ac— I W I A Q A L R $r8 I G D Q F A859 1590 Ac— I W Cha A Q A L R $r8 I G D A F N 860 1591 Ac— I W hhL A QA L R $r8 I G D A F N 861 1592 Ac— I W Adm A Q A L R $r8 I G D A F N 8621593 Ac— I W hCha A Q A L R $r8 I G D A F N 863 1594 Ac— I W hF A Q A LR $r8 I G D A F N 864 1595 Ac— I W Igl A Q A L R $r8 I G D A F N 8651596 Ac— I W F4CF3 A Q A L R $r8 I G D A F N 866 1597 Ac— I W F4tBu A QA L R $r8 I G D A F N 867 1598 Ac— I W 2Nal A Q A L R $r8 I G D A F N868 1599 Ac— I W Bip A Q A L R $r8 I G D A F N 869 1600 Ac— I W I A Q ACha R $r8 I G D A F N 870 1601 Ac— I W I A Q A hhL R $r8 I G D A F N 8711602 Ac— I W I A Q A Adm R $r8 I G D A F N 872 1603 Ac— I W I A Q A hChaR $r8 I G D A F N 873 1604 Ac— I W I A Q A hAdm R $r8 I G D A F N 8741605 Ac— I W I A Q A hF R $r8 I G D A F N 875 1606 Ac— I W I A Q A Igl R$r8 I G D A F N 876 1607 Ac— I W I A Q A F4CF3 R $r8 I G D A F N 8771608 Ac— I W I A Q A F4tBu R $r8 I G D A F N 878 1609 Ac— I W I A Q A2Nal R $r8 I G D A F N 879 1610 Ac— I W I A Q A Bip R $r8 I G D A F N880 1611 Ac— I W I A Q A L R $r8 Cba G D A F N 881 1612 Ac— I W I A Q AL R $r8 hL G D A F N 882 1613 Ac— I W I A Q A L R $r8 Cha G D A F N 8831614 Ac— I W I A Q A L R $r8 Tba G D A F N 884 1615 Ac— I W I A Q A L R$r8 hhL G D A F N 885 1616 Ac— I AmW I A Q A L R $r8 I G D A F N 8861617 Ac— I Aib I A Q A L R $r8 I G D A F N 887 1618 Ac— AmL W I A Q A LR $r8 I G D A F N 888 1619 Ac— I W AmL A Q A L R $r8 I G D A F N 8891620 Ac— I W I Aib Q A L R $r8 I G AmD A F N 890 1621 Ac— I W I A Aib AL R $r8 I G D A F N 891 1622 Ac— I W I A Q A L R $r8 I G AmD A F N 8921623 Ac— I W I A Q A L R $r8 I G D A F N 896 1624 Ac— I W Tba A Q A L R$r8 I G D A F N 897 1625 Ac— I W hL A Q A L R $r8 I G D A F N 898 1626Ac— I W Chg A Q A L R $r8 I G D A F N 899 1627 Ac— I W Ac6c A Q A L R$r8 I G D A F N 900 1628 Ac— I W Ac5c A Q A L R $r8 I G D A F N 901 1629Ac— E W I A A A L R $r8 I G D A F N 902 1630 Ac— R W I A A A L R $r8 I GD A F N 903 1631 Ac— K W I A A A L R $r8 I G D A F N 904 1632 Ac— H W IA A A L R $r8 I G D A F N 905 1633 Ac— S W I A A A L R $r8 I G D A F N906 1634 Ac— Q W I A A A L R $r8 I G D A F N 907 1635 Ac— A W I A A A LR $r8 I G D A F N 908 1636 Ac— Aib W I A A A L R $r8 I G D A F N 9091637 Ac— F W I A A A L R $r8 I G D A F N 910 1638 Ac— I D I A A A L R$r8 I G D A F N 911 1639 Ac— I R I A A A L R $r8 I G D A F N 912 1640Ac— I H I A A A L R $r8 I G D A F N 913 1641 Ac— I S I A A A L R $r8 I GD A F N 914 1642 Ac— I N I A A A L R $r8 I G D A F N 915 1643 Ac— I L IA A A L R $r8 I G D A F N 916 1644 Ac— I F I A A A L R $r8 I G D A F N917 1645 Ac— I 2Nal I A A A L R $r8 I G D A F N 918 1646 Ac— I W I S A AL R $r8 I G D A F N 919 1647 Ac— I W I L A A L R $r8 I G D A F N 9201648 Ac— I W I F A A L R $r8 I G D A F N 921 1649 Ac— I W I A L A L R$r8 I G D A F N 922 1650 Ac— I W I A A A L K $r8 I G D A F N 923 1651Ac— I W I A A A L R $r8 I Abu D A F N 924 1652 Ac— I W I A A A L R $r8 IV D A F N 925 1653 Ac— I W I A A A L R $r8 I G E A F N 926 1654 Ac— I WI A A A L R $r8 I G D A G N 927 1655 Ac— I W I A Q A L R $r8 I G D A W N928 1656 Ac— I W I A Q A L R $r8 I G D A hF N 929 1657 Ac— I W I A Q A LR $r8 I G D A F4CF3 N 930 1658 Ac— I W I A Q A L R $r8 I G D A F4tBu N931 1659 Ac— I W I A Q A L R $r8 I G D A 2Nal N 932 1660 Ac— I W I A Q AL R $r8 I G D A Bip N 933 1661 Ac— I W I A A A L R $r8 I G D A F D 9341662 Ac— I W I A A A L R $r8 I G D A F E 935 1663 Ac— I W I A A A L R$r8 I G D A F Q 936 1664 Ac— I W I A A A L R $r8 I G D A F S 937 1665Ac— I W I A A A L R $r8 I G D A F H 938 1666 Ac— I W I A A A L R $r8 I GD A F N 939 1667 Ac— I W I A Q A L R $r8 I G D A F N 940 1668 Ac— I W IA Q A L R $r8 I G D A F N 941 1669 Ac— I W I A Q A L R $r8 I G D A F N942 1670 Ac— I W I A Q A L R $r8 I G D A F N 943 1671 Ac— I W I A Q A LR $r8 I G D A F N 944 1672 Ac— I W I A Q A L R $r8 I G D A F N 945 1673Ac— I W I A A A L R $r8 I G D A F N 946 1674 Ac— I W I A A A L R $r8 I GD A F N 947 1675 Ac— I W I A A A L R $r8 I G D A F N 948 1676 Ac— I W IA A A L R $r8 I G D A F N 949 1677 Ac— I W I A A A L R $r8 I G D A F N950 1678 Ac— I W I A A A L R $r8 I G D A F N 951 1679 Ac— I W I A A A LR $r8 I G D A F N 952 1680 Ac— I W I A Q A AmL R $r8 I G D A F N 9531681 Ac— I W I A Q A L R $r8 I G AmD A F N 954 1682 Ac— I W I A Q A L R$r8 I G D A F N 955 1683 Ac— I W I A Q A L R $r8 I G D A F N 956 1684Ac— I W I A Q A A Cit $r8 I G D A F N 957 1685 Ac— I W I A Q A L Cit $r8I G N A F N 958 1686 Ac— I W I A Q A L Cit $r8 I G D A A N 959 1687 Ac—I W I A Q A L Cit $r8 I G D A V N 960 1688 Ac— I W I A Q A L R $r8 I G DA F N 961 1689 Ac— I W I A Q A L R $r8 hL G D A F N 962 1690 Ac— I W I AQ A L R $r8 hL G D A F N 963 1691 Ac— I W I A Q A L R $r8 hL G D A F N964 1692 Ac— A W I A A A L R $r8 hL G D A F N 965 1693 Ac— A W I A A A LR $r8 hL G D A F N 966 1694 Ac— I W I A Q A A R $r8 hL G D A F N 8931695 Ac— I $r8 I A Q A L R St I G D E F N 894 1696 Ac— I W I A $ A L RSt I G D E F N 895 1697 Ac— I W I A Q A L R $r8 I G D E F N SP# 16 17 1819 20 21 Calc (M + 2)/2 Found Mass LP2 Ac— A Y Y A R R —NH₂ 808 Ac— $ YY A R R —NH₂ 809 Ac— $ Y Y A R R —NH₂ 1344.74 1345.7 810 Ac— $ Y Y A R R—NH₂ 1373.75 1373.56 812 Ac— $ Y Y A —NH₂ 1103.1 1103.12 813 Ac— $ Y Y A—NH₂ 988.55 988.45 814 Ac— $ Y A A —NH₂ 964.04 963.94 815 Ac— $ Y Y A—NH₂ 1159.64 1159.87 816 Ac— $ Y Y A —NH₂ 1103.1 1102.94 817 Ac— $ Y Y A—NH₂ 1010.06 1009.9 818 Ac— $ Y Y A —NH₂ 981.55 981.86 819 Ac— $ Y Y A—NH₂ 967.53 967.45 820 Ac— $ Y Y A —NH₂ 1017.07 1016.93 821 Ac— $ Y Y A—NH₂ 972.04 971.89 822 Ac— $ A Y A —NH₂ 964.04 963.94 823 Ac— $ Y Y A—NH₂ 1185.17 1185.61 824 Ac— % Y Y A —NH₂ 1160.14 1161.28 825 Ac— $ Y YA —NH₂ 1167.14 1168.2 826 Ac— $ Y Y A —NH₂ 1150.13 1151.09 827 FITC— Ba$ Y Y A —NH₂ 1368.67 1369.79 828 5-FAM— Ba $ Y Y A —NH₂ 1353.18 1354.13829 5-FAM— Ba $ Y Y A —NH₂ 1382.18 1382.99 830 Ac— $ Y Y A —NH₂ 1102.121103.17 831 Ac— $ Y Y A —NH₂ 1188.64 1189.57 832 Ac— $ Y Y A —NH₂1188.15 1189.1 833 Ac— $ Y Y A —NH₂ 1160.13 1161.17 834 Ac— $ Y Y A —NH₂1159.64 1160.34 835 Ac— $ Y Y A —NH₂ 1131.13 1132.12 836 Ac— $ Y Y A—NH₂ 1138.14 1139.15 837 Ac— $ Y Y A —NH₂ 1160.13 1160.98 838 Ac— $ Y YA —NH₂ 1188.64 1189.66 839 Ac— $ Y Y A —NH₂ 1150.12 1151.09 840 Ac— $ YY A —NH₂ 1178.63 1179.67 841 Ac— $ Y Y A —NH₂ 1145.62 1146.55 842 Ac— $Y Y A —NH₂ 1174.13 1175.14 843 Ac— $ Y Y A —NH₂ 1121.62 1122.5 844 Ac— $Y Y A —NH₂ 1142.65 1143.59 845 Ac— $ Y Y A —NH₂ 1171.16 1171.9 846 Ac— $Y Y A —NH₂ 1100.6 1101.5 847 Ac— $ Y Y A —NH₂ 1166.65 1167.83 848 Ac— $Y Y A —NH₂ 1195.16 1196.23 849 Ac— $ Y Y A —NH₂ 1138.62 1139.61 850 Ac—$ Y Y A —NH₂ 1167.13 1168.11 851 Ac— $ Y Y A —NH₂ 1176.63 1177.63 852Ac— $ Y Y A —NH₂ 1205.14 1205.94 853 Ac— $ Y Y A —NH₂ 1176.63 1177.63854 Ac— $ Y Y A —NH₂ 1205.14 1206.13 855 Ac— $ Y Y A —NH₂ 1117.111118.15 856 Ac— $ Y Y A —NH₂ 1159.15 1159.63 857 Ac— $ Y Y A —NH₂1138.62 1139.2 858 Ac— $ Y Y A —NH₂ 1166.65 1167.3 859 Ac— $ Y Y A —NH₂1179.65 1180.15 860 Ac— $ Y Y A —NH₂ 1173.65 1174.39 861 Ac— $ Y Y A—NH₂ 1198.66 1199.28 862 Ac— $ Y Y A —NH₂ 1186.66 1186.98 863 Ac— $ Y YA —NH₂ 1183.64 1184.48 864 Ac— $ Y Y A —NH₂ 1190.65 1190.41 865 Ac— $ YY A —NH₂ 1210.62 1211.31 866 Ac— $ Y Y A —NH₂ 1204.66 1205.39 867 Ac— $Y Y A —NH₂ 1201.64 1202.2 868 Ac— $ Y Y A —NH₂ 1214.65 1215.43 869 Ac— $Y Y A —NH₂ 1179.65 1180.22 870 Ac— $ Y Y A —NH₂ 1173.65 1174.4 871 Ac— $Y Y A —NH₂ 1198.66 1199.05 872 Ac— $ Y Y A —NH₂ 1186.66 1187.25 873 Ac—$ Y Y A —NH₂ 1205.67 1206.4 874 Ac— $ Y Y A —NH₂ 1183.64 1184.29 875 Ac—$ Y Y A —NH₂ 1190.65 1190.4 876 Ac— $ Y Y A —NH₂ 1210.62 1210.94 877 Ac—$ Y Y A —NH₂ 1204.66 1205.29 878 Ac— $ Y Y A —NH₂ 1201.64 1202.15 879Ac— $ Y Y A —NH₂ 1214.65 1214.91 880 Ac— $ Y Y A —NH₂ 1165.64 1166.07881 Ac— $ Y Y A —NH₂ 1166.65 1167.37 882 Ac— $ Y Y A —NH₂ 1179.651180.22 883 Ac— $ Y Y A —NH₂ 1166.65 1167.18 884 Ac— $ Y Y A —NH₂1173.65 1173.93 885 Ac— $ Y Y A —NH₂ 1166.65 1167.18 886 Ac— $ Y Y A—NH₂ 1109.13 1109.46 887 Ac— $ Y Y A —NH₂ 1166.65 1167.27 888 Ac— $ Y YA —NH₂ 1166.65 1137.37 889 Ac— $ Y Y A —NH₂ 1173.65 1173.93 890 Ac— $ YY A —NH₂ 1138.14 1138.32 891 Ac— $ Y Y A —NH₂ 1166.65 1167.37 892 Ac— $Y F4F A —NH₂ 1160.64 1161.45 896 Ac— $ Y Y A —NH₂ 1166.65 1167.37 897Ac— $ Y Y A —NH₂ 1166.65 1167.37 898 Ac— $ Y Y A —NH₂ 1172.65 1173.47899 Ac— $ Y Y A —NH₂ 1165.64 1166.44 900 Ac— $ Y Y A —NH₂ 1158.631159.32 901 Ac— $ Y Y A —NH₂ 1139.11 1139.52 902 Ac— $ Y Y A —NH₂1152.64 1153.49 903 Ac— $ Y Y A —NH₂ 1138.63 1138.97 904 Ac— $ Y Y A—NH₂ 1143.12 1143.87 905 Ac— $ Y Y A —NH₂ 1118.1 1118.8 906 Ac— $ Y Y A—NH₂ 1138.62 1139.24 907 Ac— $ Y Y A —NH₂ 1110.1 1110.75 908 Ac— $ Y Y A—NH₂ 1117.11 1117.78 909 Ac— $ Y Y A —NH₂ 1148.12 1148.96 910 Ac— $ Y YA —NH₂ 1095.6 1096.32 911 Ac— $ Y Y A —NH₂ 1116.14 1116.95 912 Ac— $ Y YA —NH₂ 1106.62 1107.24 913 Ac— $ Y Y A —NH₂ 1081.6 1181.98 914 Ac— $ Y YA —NH₂ 1095.11 1095.58 915 Ac— $ Y Y A —NH₂ 1094.63 1095.3 916 Ac— $ Y YA —NH₂ 1111.62 1112.33 917 Ac— $ Y Y A —NH₂ 1136.63 1137.3 918 Ac— $ Y YA —NH₂ 1139.13 1139.89 919 Ac— $ Y Y A —NH₂ 1152.15 1152.94 920 Ac— $ YY A —NH₂ 1169.14 1169.86 921 Ac— $ Y Y A —NH₂ 1152.15 1152.84 922 Ac— $Y Y A —NH₂ 1117.13 1117.97 923 Ac— $ Y Y A —NH₂ 1145.14 1145.9 924 Ac— $Y Y A —NH₂ 1152.15 1152.94 925 Ac— $ Y Y A —NH₂ 1138.14 1138.87 926 Ac—$ Y Y A —NH₂ 1086.1 1086.89 927 Ac— $ Y Y A —NH₂ 1179.14 1180.04 928 Ac—$ Y Y A —NH₂ 1166.65 1167.46 929 Ac— $ Y Y A —NH₂ 1193.63 1194.38 930Ac— $ Y Y A —NH₂ 1187.67 1188.36 931 Ac— $ Y Y A —NH₂ 1184.65 1185.5 932Ac— $ Y Y A —NH₂ 1197.65 1198.54 933 Ac— $ Y Y A —NH₂ 1131.62 1132.4 934Ac— $ Y Y A —NH₂ 1138.63 1139.02 935 Ac— $ Y Y A —NH₂ 1138.14 1138.84936 Ac— $ Y Y A —NH₂ 1117.62 1118.5 937 Ac— $ Y Y A —NH₂ 1142.64 1143.25938 Ac— $ L Y A —NH₂ 1106.14 1107.05 939 Ac— $ Y A A —NH₂ 1113.631114.27 940 Ac— $ Y L A —NH₂ 1134.65 1135.33 941 Ac— $ Y Cha A —NH₂1154.66 1155.31 942 Ac— $ Y hF A —NH₂ 1158.65 1159.5 943 Ac— $ Y W A—NH₂ 1171.15 1171.78 944 Ac— $ Y 2Nal A —NH₂ 1176.65 1177 945 Ac— $ Y YD —NH₂ 1153.12 1153.77 946 Ac— $ Y Y E —NH₂ 1160.13 1160.8 947 Ac— $ Y YQ —NH₂ 1159.64 1160.26 948 Ac— $ Y Y S —NH₂ 1139.13 1139.47 949 Ac— $ YY H —NH₂ 1164.14 1165.05 950 Ac— $ Y Y R —NH₂ 1173.66 1174.4 951 Ac— $ YY K —NH₂ 1159.66 1160.26 952 Ac— $ Y Y A —NH₂ 1166.65 1167.18 953 Ac— $Y Y A —NH₂ 1166.65 1167.46 954 Ac— $ F4F Y A —NH₂ 1160.64 1161.26 955Ac— $ Y Y Aib —NH₂ 1166.65 1167.46 956 Ac— $ Y Y A —NH₂ 1139.11 1139.71957 Ac— $ Y Y A —NH₂ 1159.64 1160.4 958 Ac— $ Y Y A —NH₂ 1122.12 1122.87959 Ac— $ Y Y A —NH₂ 1136.13 1136.47 960 Ac— $ A Y A —NH₂ 1113.63 1113.9961 Ac— $ F4F Y A —NH₂ 1167.64 1168.57 962 Ac— $ Y F4F A —NH₂ 1167.641168.2 963 Ac— $ F4F F4F A —NH₂ 1168.64 1169.59 964 Ac— $ Y F4F A —NH₂1118.11 1118.89 965 Ac— $ A F4F A —NH₂ 1072.1 1072.92 966 Ac— $ F4F F4FA —NH₂ 1147.62 1148.59 893 Ac— $s8 Y Y A —NH₂ 1199.18 1199.74 894 Ac—$s8 Y Y A —NH₂ 1207.17 1207.7 895 Ac— St Y Y A $r5 A —NH₂ 1306.721307.42

TABLE 4b Selected peptidomimetic macrocycles derived from the MCL-1/BCL-XL/BC_(L)-2-binding helix of BIM. Raji Cell RT Ala IC₅₀ (nM)Viability EC₅₀ SP# Ch L VH (min)* (%) MCL-1 BCL-X_(L) (μM)** 810 0 2118.9 9.07 9.5 ND 9.2 >30 809 1 21 16.5 10.56 14.3 10.6 3.9 >30 815 0 199.1 15.07 21 8.4 22.4 6.6 962 0 19 8.3 17.69 21 27.0 13.0 0.7 *SeeExample 11 table **5% serum, 48 hr Ch = net charge; L = length in aminoacids; VH = von Heijne; RT = retention time; Ala = alanine content

Preparation of Peptidomimetic Macrocycles

Peptidomimetic macrocycles can be prepared by any of a variety ofmethods known in the art. For example, any of the residues indicated by“$” or “$r8” in Table 1, Table 1a, Table 1b, or Table 1c can besubstituted with a residue capable of forming a crosslinker with asecond residue in the same molecule or a precursor of such a residue.

Various methods to effect formation of peptidomimetic macrocycles areknown in the art. For example, the preparation of peptidomimeticmacrocycles of Formula I is described in Schafineister et al., J. Am.Chem. Soc. 122:5891-5892 (2000); Schafmeister & Verdine. J. Am. Chem.Soc. 122:5891 (2005); Walensky et al., Science 305:1466-1470 (2004);U.S. Pat. No. 7,192,713 and PCT application WO 2008/121767. Theα,α-disubstituted amino acids and amino acid precursors disclosed in thecited references can be employed in synthesis of the peptidomimeticmacrocycle precursor polypeptides. For example, the “S5-olefin aminoacid” is (S)-α-(2′-pentenyl) alanine and the “R8 olefin amino acid” is(R)-α-(2′-octenyl) alanine. Following incorporation of such amino acidsinto precursor polypeptides, the terminal olefins are reacted with ametathesis catalyst, leading to the formation of the peptidomimeticmacrocycle. In various embodiments, the following amino acids can beemployed in the synthesis of the peptidomimetic macrocycle:

In other embodiments, the peptidomimetic macrocycles are of Formula IVor IVa. Methods for the preparation of such macrocycles are described,for example, in U.S. Pat. No. 7,202,332.

Additional methods of forming peptidomimetic macrocycles which areenvisioned as suitable include those disclosed by Mustapa, M. FirouzMohd et al., J. Org. Chem (2003), 68, pp. 8193-8198: Yang, Bin et al.Bioorg Med. Chem. Lett. (2004), 14, pp. 1403-1406: U.S. Pat. Nos.5,364,851; 5,446,128; 5,824,483; 6,713,280; and 7,202,332. In suchembodiments, amino acid precursors are used containing an additionalsubstituent R- at the alpha position. Such amino acids are incorporatedinto the macrocycle precursor at the desired positions, which can be atthe positions where the crosslinker is substituted or, alternatively,elsewhere in the sequence of the macrocycle precursor. Cyclization ofthe precursor is then effected according to the indicated method.

Assays

The properties of peptidomimetic macrocycles are assayed, for example,by using the methods described below. In some embodiments, apeptidomimetic macrocycle has improved biological properties relative toa corresponding polypeptide lacking the substituents described herein.

A peptidomimetic macrocycle with enhanced cell penetrability can beprepared based on one or more properties of the polypeptide. In someembodiments, a peptidomimetic macrocycle with enhanced cellpenetrability can be prepared based on a length of the amino acidsequence of the polypeptide. In some embodiments, a peptidomimeticmacrocycle with enhanced cell penetrability can be prepared based on avon Heijne value of the polypeptide. In some embodiments, apeptidomimetic macrocycle with enhanced cell penetrability can beprepared based on a net charge carried by the polypeptide.

In some embodiments, a peptidomimetic macrocycle with enhanced cellpenetrability can be prepared based on an alanine content in the aminoacid sequence of the polypeptide. In some embodiments, a peptidomimeticmacrocycle with enhanced cell penetrability can be prepared based on anamphipathicity of the polypeptide. In some embodiments, a peptidomimeticmacrocycle with enhanced cell penetrability can be prepared based on asolubility of the polypeptide. In some embodiments, a peptidomimeticmacrocycle with enhanced cell penetrability can be prepared based on areverse-phase HPLC retention time of the polypeptide. In someembodiments, a peptidomimetic macrocycle with enhanced cellpenetrability can be prepared based on a length of the amino acidsequence of the polypeptide, a von Heijne value of the polypeptide, anet charge carried by the polypeptide, an alanine content in the aminoacid sequence of the polypeptide, an amphipathicity of the polypeptide,a solubility of the polypeptide, a reverse-phase HPLC retention time ofthe polypeptide, or any combination thereof.

A peptidomimetic macrocycle with enhanced cell penetrability can beprepared based on the length of the polypeptide. In some embodiments,the length of the prepared peptidomimetic macrocycle ranges from 10-24amino acids. For example, the length of the prepared peptidomimeticmacrocycle is 10 amino acids, 11 amino acids, 12 amino acids, 13 aminoacids, 14 amino acids, 15 amino acids, 16 amino acids, 17 amino acids,18 amino acids, 19 amino acids, 20 amino acids, 21 amino acids, 22 aminoacids, 23 amino acids, or 24 amino acids. For example, the length of theprepared peptidomimetic macrocycle ranges from 10-23, 10-22, 10-21,10-20, 10-19, 10-18, 10-17, 10-16, 10-15, 10-14, 10-13, or 10-12 aminoacids. For example, the length of the prepared peptidomimetic macrocycleranges from 1-24, 12-24, 13-24, 14-24, 15-24, 16-24, 17-24, 18-24,19-24, 20-24, 21-24, or 22-24 amino acids. In some embodiments, thelength of the prepared peptidomimetic macrocycle ranges from 11 aminoacids to 23 amino acids. For example, the length of the preparedpeptidomimetic macrocycle ranges from 11-22, 11-21, 11-20, 11-19, 11-18,11-17, 11-16, 11-15, 11-14, or 11-13 amino acids. For example, thelength of the prepared peptidomimetic macrocycle ranges from 12-23,13-23, 14-23, 15-23, 16-23, 17-23, 18-23, 19-23, 20-23, or 21-23 aminoacids. In some embodiments, the length of the prepared peptidomimeticmacrocycle ranges from 12 amino acids to 22 amino acids. For example,the length of the prepared peptidomimetic macrocycle ranges from 12-21,12-20, 12-19, 12-18, 12-17, 12-16, 12-15, or 12-14 amino acids.

For example, the length of the prepared peptidomimetic macrocycle rangesfrom 13-22, 14-22, 15-22, 16-22, 17-22, 18-22, 19-22, or 20-22 aminoacids. In some embodiments, the length of the prepared peptidomimeticmacrocycle ranges from 13 amino acids to 21 amino acids. For example,the length of the prepared peptidomimetic macrocycle ranges from 13-20,13-19, 13-18, 13-17, 13-16, or 13-15 amino acids. For example, thelength of the prepared peptidomimetic macrocycle ranges from 14-21,15-21, 16-21, 17-21, 18-21, or 19-21 amino acids. In some embodiments,the length of the prepared peptidomimetic macrocycle ranges from 14amino acids to 20 amino acids. For example, the length of the preparedpeptidomimetic macrocycle ranges from −19, 14-18, 14-17, or 14-16 aminoacids. For example, the length of the prepared peptidomimetic macrocycleranges from 15-20, 16-20, 17-20, or 18-20 amino acids. In someembodiments, the length of the prepared peptidomimetic macrocycle rangesfrom 15 amino acids to 19 amino acids. For example, the length of theprepared peptidomimetic macrocycle ranges from 15-18 or 15-17 aminoacids. For example, the length of the prepared peptidomimetic macrocycleranges from 16-19 or 17-19 amino acids. In some embodiments, the lengthof the prepared peptidomimetic macrocycle ranges from 16 amino acids to18 amino acids. For example, the length of the prepared peptidomimeticmacrocycle is 17. In some embodiments, the length of the preparedpeptidomimetic macrocycle is 14. In some embodiments, the length of theprepared peptidomimetic macrocycle is 15. In some embodiments, thelength of the prepared peptidomimetic macrocycle is 16. In someembodiments, the length of the prepared peptidomimetic macrocycle is 17.In some embodiments, the length of the prepared peptidomimeticmacrocycle is 18. In some embodiments, the length of the preparedpeptidomimetic macrocycle is 19. In some embodiments, the length of theprepared peptidomimetic macrocycle is 20. In some embodiments, thelength of the prepared peptidomimetic macrocycle is 21.

A peptidomimetic macrocycle with enhanced cell penetrability can beprepared based on the von Heijne value of the polypeptide. In someembodiments, the von Heijne value of the prepared peptidomimeticmacrocycle ranges from 2 to 9. For example, the von Heijne value of theprepared peptidomimetic macrocycle is 2, 3, 4, 5, 6, 7, 8, or 9, alongwith all values in between. For example, the von Heijne value of theprepared peptidomimetic macrocycle ranges from 2-8, 2-7, 2-6, 2-5, 2-4,or 2-3. For example, the von Heijne value of the prepared peptidomimeticmacrocycle ranges from 3-9, 4-9, 5-9, 6-9, 7-9, or 8-9. In someembodiments, the von Heijne value of the prepared peptidomimeticmacrocycle ranges from 3 to 8. For example, the von Heijne value of theprepared peptidomimetic macrocycle ranges from 3-7, 3-6, 3-5, or 3-4.For example, the von Heijne value of the prepared peptidomimeticmacrocycle ranges from 4-8, 5-8, 6-8, or 7-9.

In some embodiments, the von Heijne value of the prepared peptidomimeticmacrocycle ranges from 4 to 7. For example, the von Heijne value of theprepared peptidomimetic macrocycle ranges from 4-6 or 4-5. For example,the von Heijne value of the prepared peptidomimetic macrocycle rangesfrom 5-7 or 5-6. In some embodiments, the von Heijne value of theprepared peptidomimetic macrocycle ranges from 4-6. In some embodiments,the von Heijne value of the prepared peptidomimetic macrocycle rangesfrom 4-5. In some embodiments, the von Heijne value of the preparedpeptidomimetic macrocycle ranges from 4.5-5.5, including 4.5, 4.6, 4.7,4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, and 5.5 along with all values inbetween. In some embodiments, the von Heijne value of the preparedpeptidomimetic macrocycle ranges from 4.5-9.5. For example, the vonHeijne value of the prepared peptidomimetic macrocycle ranges from4.5-8.5, 4.5-7.5, 4.5-6.5, 5.5-9.5, 5.5-8.5, 5.5-7.5, 5.5-6.5, 6.5-9.5,6.5-8.5, 6.5-7.5, 7.5-9.5, or 7.5-8.5.

A peptidomimetic macrocycle with enhanced cell penetrability can beprepared based on the net charge carried by a peptide. For example,peptidomimetic macrocycles carrying a high number of negative chargescan have poor cell permeability. A peptidomimetic macrocycle carrying ahigh number of positive charges can have good cell permeability, but cancause undesired cell damage (e.g., cell lysis). A preparedpeptidomimetic macrocycle can carry a net charge such that thepolypeptide is cell permeable, but not damaging to cells (e.g., does notcause cell lysis). In some embodiments, the net charge of the preparedpeptidomimetic macrocycle ranges from −4 to +2, including −4, −3, −2,−1, 0, +1, and +2. In some embodiments, the net charge of the preparedpeptidomimetic macrocycle ranges from −3 to +1, including −3, −2, −1, 0and +1.

In some embodiments, the net charge of the prepared peptidomimeticmacrocycle ranges from −2 to 0, including −2, −1, and 0. In someembodiments, the net charge of the prepared peptidomimetic macrocycle iszero or negative. In some embodiments, the net charge of the preparedpeptidomimetic macrocycle is not positive. In some embodiments, the netcharge of the prepared peptidomimetic macrocycle is zero or is notpositive. In some embodiments, the net charge of the preparedpeptidomimetic macrocycle is −2. In some embodiments, the net charge ofthe prepared peptidomimetic macrocycle is −1. In some embodiments, thenet charge of the prepared peptidomimetic macrocycle is 0.

A peptidomimetic macrocycle with enhanced cell penetrability can beprepared based on the alanine content of the polypeptide. In someembodiments, the alanine content of the prepared peptidomimeticmacrocycle ranges from 15% to 50%. For example, the alanine content ofthe prepared peptidomimetic macrocycle can be 15%, 16%, 17%, 18%, 19%,20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%,34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%,48%, 49%, and 50%, along with all values in between. In someembodiments, the alanine content of the prepared peptidomimeticmacrocycle ranges from 15% to 45%. In some embodiments, the alaninecontent of the prepared peptidomimetic macrocycle ranges from 15% to40%. In some embodiments, the alanine content of the preparedpeptidomimetic macrocycle ranges from 15% to 35%. In some embodiments,the alanine content of the prepared peptidomimetic macrocycle rangesfrom 20% to 50%. In some embodiments. the alanine content of theprepared peptidomimetic macrocycle ranges from 20% to 45%. In someembodiments, the alanine content of the prepared peptidomimeticmacrocycle ranges from 20% to 40%. In some embodiments, the alaninecontent of the prepared peptidomimetic macrocycle ranges from 20% to35%. In some embodiments, the alanine content of the preparedpeptidomimetic macrocycle ranges from 20% to 30%. In some embodiments,the alanine content of the prepared peptidomimetic macrocycle rangesfrom 20% to 25%. In some embodiments, the alanine content of theprepared peptidomimetic macrocycle ranges from 25% to 50%. In someembodiments, the alanine content of the prepared peptidomimeticmacrocycle ranges from 25% to 45%. In some embodiments, the alaninecontent of the prepared peptidomimetic macrocycle ranges from 25% to40%. For example, the alanine content of the prepared peptidomimeticmacrocycle can be 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%,36%, 37%, 38%, 39%, and 40% along with all values in between. In someembodiments, the alanine content of the prepared peptidomimeticmacrocycle ranges from 25% to 35%. In some embodiments, the alaninecontent of the prepared peptidomimetic macrocycle ranges from 25% to30%. In some embodiments, the alanine content of the preparedpeptidomimetic macrocycle ranges from 30% to 50%. In some embodiments,the alanine content of the prepared peptidomimetic macrocycle rangesfrom 30% to 45%. In some embodiments, the alanine content of theprepared peptidomimetic macrocycle ranges from 30% to 40%. In someembodiments, the alanine content of the prepared peptidomimeticmacrocycle ranges from 30% to 35%. In some embodiments, the alaninecontent of the prepared peptidomimetic macrocycle ranges from 35% to50%. In some embodiments, the alanine content of the preparedpeptidomimetic macrocycle ranges from 35% to 45%. In some embodiments,the alanine content of the prepared peptidomimetic macrocycle rangesfrom 35% to 40%.

A peptidomimetic macrocycle with enhanced cell penetrability can beprepared based on the length and the von Heijne value of thepolypeptide. For example, the length of the prepared peptidomimeticmacrocycle ranges from 10 amino acids to 24 amino acids, from 11 aminoacids to 23 amino acids, from 12 amino acids to 22 amino acids, from 13amino acids to 21 amino acids, from 14 amino acids to 20 amino acids,from 15 amino acids to 19 amino acids, or from 16 amino acids to 18amino acids, and the von Heijne value of the prepared peptidomimeticmacrocycle ranges from 2 to 9, from 3 to 8, from 4 to 7, from 4 to 6, orfrom 4 to 5. For example, the length of the prepared peptidomimeticmacrocycle is 10 amino acids, 11 amino acids, 12 amino acids, 13 aminoacids, 14 amino acids, 15 amino acids, 16 amino acids, 17 amino acids,18 amino acids, 19 amino acids, 20 amino acids, or 21 amino acids, andthe von Heijne value of the prepared peptidomimetic macrocycle rangesfrom 4.5 to 5.5. For example, the prepared peptidomimetic macrocycle hasa length ranging from 14 amino acids to 20 amino acids, and a von Heijnevalue ranging from 4 and 7.

A peptidomimetic macrocycle with enhanced cell penetrability can beprepared based on the length and the alanine content of the polypeptide.For example, the length of the prepared peptidomimetic macrocycle rangesfrom 10 amino acids to 24 amino acids, from 11 amino acids to 23 aminoacids, from 12 amino acids to 22 amino acids, from 13 amino acids to 21amino acids, from 14 amino acids to 20 amino acids, from 15 amino acidsto 19 amino acids, or from 16 amino acids to 18 amino acids, and thealanine content of the prepared peptidomimetic macrocycle ranges from15% to 50%, including 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%,25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%,39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, and 50% alongwith all values in between. For example, the length of the preparedpeptidomimetic macrocycle is 10 amino acids, 11 amino acids, 12 aminoacids, 13 amino acids, 14 amino acids, 15 amino acids, 16 amino acids,17 amino acids, 18 amino acids, 19 amino acids, 20 amino acids, or 21amino acids, and the alanine content of the prepared peptidomimeticmacrocycle ranges from 25% to 40%, including 25%, 26%, 27%, 28%, 29%,30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, and 40% along with allvalues in between. For example, the prepared peptidomimetic macrocyclehas a length ranging from 14 amino acids to 20 amino acids, and analanine content ranging from 25% to 40%.

A peptidomimetic macrocycle with enhanced cell penetrability can beprepared based on the length and the net charge of the polypeptide. Forexample, the length of the prepared peptidomimetic macrocycle rangesfrom 10 amino acids to 24 amino acids, from 11 amino acids to 23 aminoacids, from 12 amino acids to 22 amino acids, from 13 amino acids to 21amino acids, from 14 amino acids to 20 amino acids, from 15 amino acidsto 19 amino acids, or from 16 amino acids to 18 amino acids, and the netcharge of the prepared peptidomimetic macrocycle ranges from −3 to 1,including −3, −2, −1, 0 and 1. For example, the length of the preparedpeptidomimetic macrocycle is 10 amino acids, 11 amino acids, 12 aminoacids, 13 amino acids, 14 amino acids, 15 amino acids, 16 amino acids,17 amino acids, 18 amino acids, 19 amino acids, 20 amino acids, or 21amino acids, and the net charge of the prepared peptidomimeticmacrocycle ranges from −3 to 1, including −3, −2, −1, 0 and 1. Forexample, the prepared peptidomimetic macrocycle has a length rangingfrom 14 amino acids to 20 amino acids, and a net charge ranging from −2to 0.

A peptidomimetic macrocycle with enhanced cell penetrability can beprepared based on the von Heijne value and the net charge of thepolypeptide. For example, the von Heijne value of the preparedpeptidomimetic macrocycle ranges from 2 to 9, from 3 to 8, from 4 to 7,from 4 to 6, or from 4 to 5, and the net charge of the preparedpeptidomimetic macrocycle ranges from −3 to 1, including -3, −2, −1, 0and 1. For example, the von Heijne value of the prepared peptidomimeticmacrocycle ranges from 4.5 to 5.5, including 4.5, 4.6, 4.7, 4.8, 4.9,5.0, 5.1, 5.2, 5.3, 5.4, and 5.5 along with all values in between, andthe net charge of the prepared peptidomimetic macrocycle ranges from −3to 1, including -3, −2, −1, 0 and 1. For example, the preparedpeptidomimetic macrocycle has a von Heijne value ranging from 4 and 7,and a net charge ranging from −2 to 0.

A peptidomimetic macrocycle with enhanced cell penetrability can beprepared based on the von Heijne value and the alanine content of thepolypeptide. For example, the von Heijne value of the preparedpeptidomimetic macrocycle ranges from 2 to 9, from 3 to 8, from 4 to 7,from 4 to 6, or from 4 to 5, and the alanine content of the preparedpeptidomimetic macrocycle ranges from 15% to 50%, including 15%, 16%,17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%,31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%,45%, 46%, 47%, 48%, 49%, and 50% along with all values in between. Forexample, the von Heijne value of the prepared peptidomimetic macrocycleranges from 4.5 to 5.5, including 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1,5.2, 5.3, 5.4, and 5.5 along with all values in between, and the alaninecontent of the prepared peptidomimetic macrocycle ranges from 25% to40%, including 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%,36%, 37%, 38%, 39%, and 40% along with all values in between. Forexample, the prepared peptidomimetic macrocycle has a von Heijne valueranging from 4 and 7, and an alanine content ranging from 25% to 40%.

A peptidomimetic macrocycle with enhanced cell penetrability can beprepared based on length, von Heijne value and alanine content of thepolypeptide. For example, the prepared peptidomimetic macrocycle has alength ranging from 14 amino acids to 20 amino acids, a von Heijne valueranging from 4 and 7, and an alanine content ranging from 25% to 40%.

A peptidomimetic macrocycle with enhanced cell penetrability can beprepared based on the von Heijne value and the net charge of thepolypeptide. For example, the prepared peptidomimetic macrocycle has alength ranging from 14 amino acids to 20 amino acids, a von Heijne valueranging from 4 and 7, and a net charge ranging from −2 to 0.

A peptidomimetic macrocycle with enhanced cell penetrability can b^(e)prepared based on the von Heijne value, the net charge, and the alaninecontent of the polypeptide. For example, the prepared peptidomimeticmacrocycle has a von Heijne value ranging from 4 and 7. a net chargeranging from −2 to 0, and an alanine content ranging from 25% to 40%.

A peptidomimetic macrocycle with enhanced cell penetrability can beprepared based on the length, the net charge, and the alanine content ofthe polypeptide. For example, the prepared peptidomimetic macrocycle hasa length ranging from 14 amino acids to 20 amino acids, a net chargeranging from −2 to 0, and an alanine content ranging from 25% to 40%.

A peptidomimetic macrocycle with enhanced cell penetrability can beprepared based on the length of its amino acid sequence, its von Heijnevalue, its net charge, and the alanine content of its amino acidsequence. For example, the prepared peptidomimetic macrocycle has alength ranging from 14 amino acids to 20 amino acids, a von Heijne valueranging from 4 and 7, a net charge ranging from −2 to 0, and an alaninecontent ranging from 25% to 40%.

In some embodiments, a peptidomimetic macrocycle with enhanced cellpenetrability can be prepared based on the reverse-phase HPLC retentiontime of the polypeptide.

In some embodiments, a peptidomimetic macrocycle with enhanced cellpenetrability can be prepared based on amphipathicity of thepolypeptide.

In some embodiments, a peptidomimetic macrocycle with enhanced cellpenetrability can be prepared based on solubility of the polypeptide,for example if the prepared peptidomimetic macrocycle is determined tobe soluble based on visual examination of the turbidity of a solution ofthe polypeptide.

Assay to Determine α-Helicity

In solution, the secondary structure of polypeptides with α-helicaldomains will reach a dynamic equilibrium between random coil structuresand α-helical structures, often expressed as a “percent helicity”. Thus,for example, alpha-helical domains are predominantly random coils insolution, with α-helical content usually under 25%. Peptidomimeticmacrocycles with optimized linkers, on the other hand, possess, forexample, an alpha-helicity that is at least two-fold greater than thatof a corresponding uncrosslinked polypeptide. In some embodiments,macrocycles will possess an alpha-helicity of greater than 50%. To assaythe helicity of peptidomimetic macrocycles, the compounds are dissolvedin an aqueous solution (e.g. 50 mM potassium phosphate solution at pH 7,or distilled H₂O, to concentrations of 25-50 μM). Circular dichroism(CD) spectra are obtained on a spectropolarimeter (e.g., Jasco J-710)using standard measurement parameters (e.g. temperature, 20° C.;wavelength, 190-260 nm; step resolution, 0.5 nm; speed, 20 nm/sec;accumulations, 10; response, 1 sec; bandwidth, 1 nm; path length, 0.1cm). The α-helical content of each peptide is calculated by dividing themean residue ellipticity (e.g. [Φ]222obs) by the reported value for amodel helical decapeptide (Yang et al. (1986), Methods Enzymol.130:208)).

Assay to Determine Melting Temperature (Tm)

A peptidomimetic macrocycle comprising a secondary structure such as anα-helix exhibits, for example, a higher melting temperature than acorresponding uncrosslinked polypeptide. Typically peptidomimeticmacrocycles exhibit Tm of >60° C. representing a highly stable structurein aqueous solutions. To assay the effect of macrocycle formation onmelting temperature, peptidomimetic macrocycles or unmodified peptidesare dissolved in distilled H₂O (e.g. at a final concentration of 50 μM)and the Tm is determined by measuring the change in ellipticity over atemperature range (e.g. 4 to 95° C.) on a spectropolarimeter (e.g.,Jasco J-710) using standard parameters (e.g. wavelength 222 nm; stepresolution, 0.5 nm; speed, 20 nm/sec; accumulations, 10; response, 1sec; bandwidth, 1 nm; temperature increase rate: 1° C./min; path length,0.1 cm).

Protease Resistance Assay

The amide bond of the peptide backbone is susceptible to hydrolysis byproteases, thereby rendering peptidic compounds vulnerable to rapiddegradation in vivo. Peptide helix formation, however, typically buriesthe amide backbone and therefore can shield it from proteolyticcleavage. The peptidomimetic macrocycles can be subjected to in vitrotrypsin proteolysis to assess for any change in degradation ratecompared to a corresponding uncrosslinked polypeptide. For example, thepeptidomimetic macrocycle and a corresponding uncrosslinked polypeptideare incubated with trypsin agarose and the reactions quenched at varioustime points by centrifugation and subsequent HPLC injection toquantitate the residual substrate by ultraviolet absorption at 280 nm.Briefly, the peptidomimetic macrocycle and peptidomimetic precursor (5mcg) are incubated with trypsin agarose (Pierce) (S/E ˜125) for 0, 10,20, 90, and 180 minutes. Reactions are quenched by tabletopcentrifugation at high speed; remaining substrate in the isolatedsupernatant is quantified by HPLC-based peak detection at 280 nm. Theproteolytic reaction displays first order kinetics and the rateconstant, k, is determined from a plot of ln[S] versus time(k=−1Xslope).

Ex Vivo Stability Assay

Peptidomimetic macrocycles with optimized linkers possess, for example,an ex vivo half-life that is at least two-fold greater than that of acorresponding uncrosslinked polypeptide, and possess an ex vivohalf-life of 12 hours or more. For ex vivo serum stability studies, avariety of assays can be used. For example, a peptidomimetic macrocycleand a corresponding uncrosslinked polypeptide (2 mcg) are incubated withfresh mouse, rat and/or human serum (2 mL) at 37° C. for 0, 1, 2, 4, 8,and 24 hours. To determine the level of intact compound, the followingprocedure can be used: The samples are extracted by transferring 100 μlof sera to 2 ml centrifuge tubes followed by the addition of 10 μL of50% formic acid and 500 μL acetonitrile and centrifugation at 14,000 RPMfor 10 min at 4+2° C. The supernatants are then transferred to fresh 2ml tubes and evaporated on Turbovap under N₂<10 psi, 37° C. The samplesare reconstituted in 100 μL of 50:50 acetonitrile:water and submitted toLC-MS/MS analysis.

In Vitro Binding Assays

To assess the binding and affinity of peptidomimetic macrocycles andpeptidomimetic precursors to acceptor proteins, a fluorescencepolarization assay (FPA) is used, for example. The FPA techniquemeasures the molecular orientation and mobility using polarized lightand fluorescent tracer. When excited with polarized light, fluorescenttracers (e.g., FITC) attached to molecules with high apparent molecularweights (e.g. FITC-labeled peptides bound to a large protein) emithigher levels of polarized fluorescence due to their slower rates ofrotation as compared to fluorescent tracers attached to smallermolecules (e.g. FITC- labeled peptides that are free in solution).

For example, fluoresceinated peptidomimetic macrocycles (25 nM) areincubated with the acceptor protein (25-1000 nM) in binding buffer (140mM NaCl, 50 mM Tris-HCL, pH 7.4) for 30 minutes at room temperature.Binding activity is measured, for example, by fluorescence polarizationon a luminescence spectrophotometer (e.g. Perkin-Elmer LS50B). Kd valuescan be determined by nonlinear regression analysis using, for example,Graphpad Prism software (GraphPad Software, Inc., San Diego, Calif.). Apeptidomimetic macrocycle shows. In some embodiments, similar or lowerKd than a corresponding uncrosslinked polypeptide.

In Vitro Displacement Assays to Characterize Antagonists ofPeptide-Protein Interactions

To assess the binding and affinity of compounds that antagonize theinteraction between a peptide and an acceptor protein, a fluorescencepolarization assay (FPA) utilizing a fluoresceinated peptidomimeticmacrocycle derived from a peptidomimetic precursor sequence is used, forexample. The FPA technique measures the molecular orientation andmobility using polarized light and fluorescent tracer. When excited withpolarized light, fluorescent tracers (e.g., FITC) attached to moleculeswith high apparent molecular weights (e.g. FITC-labeled peptides boundto a large protein) emit higher levels of polarized fluorescence due totheir slower rates of rotation as compared to fluorescent tracersattached to smaller molecules (e.g. FITC-labeled peptides that are freein solution). A compound that antagonizes the interaction between thefluoresceinated peptidomimetic macrocycle and an acceptor protein willbe detected in a competitive binding FPA experiment.

For example, putative antagonist compounds (1 nM to 1 mM) and afluoresceinated peptidomimetic macrocycle (25 nM) are incubated with theacceptor protein (50 nM) in binding buffer (140 mM NaCl, 50 mM Tris-HCL,pH 7.4) for 30 minutes at room temperature. Antagonist binding activityis measured, for example, by fluorescence polarization on a luminescencespectrophotometer (e.g. Perkin-Elmer LS50B). Kd values can be determinedby nonlinear regression analysis using, for example, Graphpad Prismsoftware (GraphPad Software, Inc., San Diego, Calif.).

Any class of molecule, such as small organic molecules, peptides,oligonucleotides or proteins can be examined as putative antagonists inthis assay.

Assay for Protein-Ligand Binding by Affinity Selection-Mass Spectrometry

To assess the binding and affinity of test compounds for proteins, anaffinity-selection mass spectrometry assay is used, for example.Protein-ligand binding experiments are conducted according to thefollowing representative procedure outlined for a system-wide controlexperiment using 1 μM peptidomimetic macrocycle plus 5 μM hMDM2. A 1 μLDMSO aliquot of a 40 μM stock solution of peptidomimetic macrocycle isdissolved in 19 μL of PBS (Phosphate-buffered saline: 50 mM, pH 7.5Phosphate buffer containing 150 mM NaCl). The resulting solution ismixed by repeated pipetting and clarified by centrifugation at 10 000 gfor 10 min. To a 4 μL aliquot of the resulting supernatant is added 4 μLof 10 μM hMDM2 in PBS. Each 8.0 μL experimental sample thus contains 40pmol (1.5 μg) of protein at 5.0 μM concentration in PBS plus 1 μMpeptidomimetic macrocycle and 2.5% DMSO. Duplicate samples thus preparedfor each concentration point are incubated for 60 min at roomtemperature, and then chilled to 4° C. prior to size-exclusionchromatography-LC-MS analysis of 5.0 μL injections. Samples containing atarget protein, protein-ligand complexes, and unbound compounds areinjected onto an SEC column, where the complexes are separated fromnon-binding component by a rapid SEC step. The SEC column eluate ismonitored using UV detectors to confirm that the early-eluting proteinfraction, which elutes in the void volume of the SEC column, is wellresolved from unbound components that are retained on the column. Afterthe peak containing the protein and protein-ligand complexes elutes fromthe primary UV detector, it enters a sample loop where it is excisedfrom the flow stream of the SEC stage and transferred directly to theLC-MS via a valving mechanism. The (M+3H)³⁺ ion of the peptidomimeticmacrocycle is observed by ESI-MS at the expected m/z, confirming thedetection of the protein-ligand complex.

Assay for Protein-Ligand Kd Titration Experiments

To assess the binding and affinity of test compounds for proteins, aprotein-ligand Kd titration experiment is performed, for example.Protein-ligand K_(e) titrations experiments are conducted as follows: 2μL DMSO aliquots of a serially diluted stock solution of titrantpeptidomimetic macrocycle (5, 2.5, . . . , 0.098 mM) are prepared thendissolved in 38 μL of PBS. The resulting solutions are mixed by repeatedpipetting and clarified by centrifugation at 10 000 g for 10 min. To 4.0μL aliquots of the resulting supernatants is added 4.0 μL of 10 μM hMDM2in PBS. Each 8.0 μL experimental sample thus contains 40 pmol (1.5 μg)of protein at 5.0 μM concentration in PBS, varying concentrations (125,62.5 . . . , 0.24 μM) of the titrant peptide, and 2.5% DMSO.

Duplicate samples thus prepared for each concentration point areincubated at room temperature for 30 min, then chilled to 4° C. prior toSEC-LC-MS analysis of 2.0 μL injections. The (M+H)¹⁺, (M+2H)²⁺,(M+3H)³⁺, and/or (M+Na)¹⁺ ion is observed by ESI-MS: extracted ionchromatograms are quantified. then fit to equations to derive thebinding affinity K_(d) as described in “A General Technique to RankProtein-Ligand Binding Affinities and Determine Allosteric vs. DirectBinding Site Competition in Compound Mixtures.” Annis, D. A.; Nazef, N.;Chuang, C. C.; Scott, M. P.; Nash, H. M. J. Am. Chem. Soc. 2004,126,15495-15503; also in “ALIS: An Affnity Selection-Mass SpectrometrySystem for the Discovery and Characterization of Protein-ligandInteractions” D. A. Annis, C.-C. Chuang, and N. Nazef. In MassSpectrometry in Medicinal Chemistry. Edited by Wanner K, Hbfner G:Wiley-VCH; 2007:121-184. Mannhold R, Kubinyi H, Folkers G (SeriesEditors): Methods and Principles in Medicinal Chemistry.

Assay for Competitive Binding Experiments by Affinity Selection-MassSpectrometry

To determine the ability of test compounds to bind competitively toproteins, an affinity selection mass spectrometry assay is performed,for example. A mixture of ligands at 40 μM per component is prepared bycombining 2 μL aliquots of 400 μM stocks of each of the three compoundswith 14 μL of DMSO. Then, 1 μL aliquots of this 40 μM per componentmixture are combined with 1 μL DMSO aliquots of a serially diluted stocksolution of titrant peptidomimetic macrocycle (10, 5, 2.5, . . . , 0.078mM). These 2 μL samples are dissolved in 38 μL of PBS. The resultingsolutions were mixed by repeated pipetting and clarified bycentrifugation at 10 000 g for 10 min. To 4.0 μL aliquots of theresulting supernatants is added 4.0 μL of 10 μM hMDM2 protein in PBS.

Each 8.0 μL experimental sample thus contains 40 pmol (1.5 μg) ofprotein at 5.0 μM concentration in PBS plus 0.5 μM ligand, 2.5% DMSO,and varying concentrations (125, 62.5 . . . , 0.98 μM) of the titrantpeptidomimetic macrocycle. Duplicate samples thus prepared for eachconcentration point are incubated at room temperature for 60 min, thenchilled to 4° C. prior to SEC-LC-MS analysis of 2.0 μL injections.Additional details on these and other methods are provided in “A GeneralTechnique to Rank Protein-Ligand Binding Affinities and DetermineAllosteric vs. Direct Binding Site Competition in Compound Mixtures.”Annis, D. A.; Nazef, N.; Chuang, C. C.; Scott, M. P.; Nash, H. M. J Am.Chem. Soc. 2004, 126, 15495-15503; also in “ALIS: An AffinitySelection-Mass Spectrometry System for the Discovery andCharacterization of Protein-Ligand Interactions” D. A. Annis, C.-C.Chuang, and N. Nazef. In Mass Spectrometry in Medicinal Chemistry.Edited by Wanner K, Höfner G: Wiley-VCH: 2007:121-184. Mannhold R,Kubinyi H. Folkers G (Series Editors): Methods and Principles inMedicinal Chemistry.

Binding Assays in Intact Cells

It is possible to measure binding of peptides or peptidomimeticmacrocycles to their natural acceptors in intact cells byimmunoprecipitation experiments. For example, intact cells are incubatedwith fluoresceinated (FITC-labeled) compounds for 4 hrs in the absenceof serum, followed by serum replacement and further incubation thatranges from 4-18 hrs. Cells are then pelleted and incubated in lysisbuffer (50 mM Tris [pH 7.6], 150 mM NaCl, 1% CHAPS and proteaseinhibitor cocktail) for 10 minutes at 4° C. Extracts are centrifuged at14,000 rpm for 15 minutes and supernatants collected and incubated with10 μl goat anti-FITC antibody for 2 hrs, rotating at 4° C. followed byfurther 2 hrs incubation at 4° C. with protein A/G Sepharose (50 μl of50% bead slurry). After quick centrifugation, the pellets are washed inlysis buffer containing increasing salt concentration (e.g., 150, 300,500 mM). The beads are then re-equilibrated at 150 mM NaCl beforeaddition of SDS-containing sample buffer and boiling. Aftercentrifugation, the supernatants are optionally electrophoresed using4%-12% gradient Bis-Tris gels followed by transfer into Immobilon-Pmembranes. After blocking, blots are optionally incubated with anantibody that detects FITC and also with one or more antibodies thatdetect proteins that bind to the peptidomimetic macrocycle.

Cellular Penetrability Assays

A peptidomimetic macrocycle is, for example, more cell penetrablecompared to a corresponding uncrosslinked macrocycle. Peptidomimeticmacrocycles with optimized linkers possess, for example, cellpenetrability that is at least two-fold greater than a correspondinguncrosslinked macrocycle, and often 20% or more of the appliedpeptidomimetic macrocycle will b^(e) observed to have penetrated thecell after 4 hours. To measure the cell penetrability of peptidomimeticmacrocycles and corresponding uncrosslinked macrocycle, intact cells areincubated with fluorescently-labeled (e.g. fluoresceinated)peptidomimetic macrocycles or corresponding uncrosslinked macrocycle (10μM) for 4 hrs in serum free media at 37° C., washed twice with media andincubated with trypsin (0.25%) for 10 min at 37° C. The cells are washedagain and resuspended in PBS. Cellular fluorescence is analyzed, forexample, by using either a FACSCalibur flow cytometer or Cellomics'KineticScan @ HCS Reader.

Cellular Efficacy Assays

The efficacy of certain peptidomimetic macrocycles is determined, forexample, in cell-based killing assays using a variety of tumorigenic andnon-tumorigenic cell lines and primary cells derived from human or mousecell populations. Cell viability is monitored, for example, over 24-96hrs of incubation with peptidomimetic macrocycles (0.5 to 50 μM) toidentify those that kill at EC₅<10 μM. Several standard assays thatmeasure cell viability are commercially available and are optionallyused to assess the efficacy of the peptidomimetic macrocycles. Inaddition, assays that measure Annexin V and caspase activation areoptionally used to assess whether the peptidomimetic macrocycles killcells by activating the apoptotic machinery. For example, the CellTiter-glo assay is used which determines cell viability as a function ofintracellular ATP concentration.

In Vivo Stability Assay

To investigate the in vivo stability of the peptidomimetic macrocycles,the compounds are, for example, administered to mice and/or rats by IV,IP, PO or inhalation routes at concentrations ranging from 0.1 to 50mg/kg and blood specimens withdrawn at 0′, 5′, 15′, 30′, 1 hr, 4 hrs, 8hrs and 24 hours post-injection. Levels of intact compound in 25 μL offresh serum are then measured by LC-MS/MS as above.

In Vivo Efficacy in Animal Models

To determine the anti-oncogenic activity of peptidomimetic macrocyclesin vivo, the compounds are, for example, given alone (IP, IV, PO, byinhalation or nasal routes) or in combination with sub-optimal doses ofrelevant chemotherapy (e.g., cyclophosphamide, doxorubicin, etoposide).In one example, 5×10⁶ RS4; 11 cells (established from the bone marrow ofa patient with acute lymphoblastic leukemia) that stably expressluciferase are injected by tail vein in NOD-SCID mice 3 hrs after theyhave been subjected to total body irradiation. If left untreated, thisform of leukemia is fatal in 3 weeks in this model. The leukemia isreadily monitored, for example, by injecting the mice with D-luciferin(60 mg/kg) and imaging the anesthetized animals (e.g., Xenogen In VivoImaging System, Caliper Life Sciences, Hopkinton, Mass.). Total bodybioluminescence is quantified by integration of photonic flux(photons/sec) by Living Image Software (Caliper Life Sciences,Hopkinton, Mass.). Peptidomimetic macrocycles alone or in combinationwith sub-optimal doses of relevant chemotherapeutics agents are, forexample, administered to leukemic mice (10 days after injection/day 1 ofexperiment, in bioluminescence range of 14-16) by tail vein or IP routesat doses ranging from 0.1 mg/kg to 50 mg/kg for 7 to 21 days.Optionally, the mice are imaged throughout the experiment every otherday and survival monitored daily for the duration of the experiment.Expired mice are optionally subjected to necropsy at the end of theexperiment. Another animal model is implantation into NOD-SCID mice ofDoHH2, a cell line derived from human follicular lymphoma, that stablyexpresses luciferase. These in vivo tests optionally generatepreliminary pharmacokinetic, pharmacodynamic and toxicology data.

Clinical Trials

To determine the suitability of the peptidomimetic macrocycles fortreatment of humans, clinical trials are performed. For example,patients diagnosed with solid tumor and in need of treatment can beselected and separated in treatment and one or more control groups,wherein the treatment group is administered a peptidomimetic macrocycle,while the control groups receive a placebo or a known anti-cancer drug.The treatment safety and efficacy of the peptidomimetic macrocycles canthus be evaluated by performing comparisons of the patient groups withrespect to factors such as survival and quality-of-life. In thisexample, the patient group treated with a peptidomimetic macrocycle canshow improved long-term survival compared to a patient control grouptreated with a placebo.

Chemical Stability

To assay the chemical stability of the aqueous pharmaceuticalformulations disclosed herein, I mL of the aqueous pharmaceuticalformulation is filled in 2-mL vials with 13-mm ç stoppers. The smallervial size can help provide a greater surface-to-volume ratio which wouldamplify any container/closure effects on product stability. To assurethat all surfaces of the vials were challenged, the vials can be storedin an inverted position. The vials are stored at the desired assaytemperature, for example −20° C. 5° C., 20° C., and 40° C. for thedesired assay time. For example for 1, 2, 3 or 6 months. The samples areanalyzed by reverse phase HPLC. Tables 8-12 and 14-17 depict the resultsof this study. The samples can also be analyzed for particulate matter.

In Vitro Testing for Inhibition of Influenza Replication

This influenza antiviral evaluation assay examines the effects ofcompounds at designated dose-response concentrations. See also Noah, J.W., W. Severson, D. L. Noah, L. Rasmussen, E. L. White, and C. B.Jonsson, Antiviral Res, 2007, 73(1): p. 50-9. Madin Darby canine kidney(MDCK) cells are used in the assay to test the efficacy of the compoundsin preventing the cytopathic effect (CPE) induced by influenzainfection. Either Ribavirin or Tamiflu is included in each run as apositive control compound. Subconfluent cultures of MDCK cells areplated into 96-well plates for the analysis of cell viability(cytotoxicity) and antiviral activity (CPE). Drugs are added to thecells 24 hr later. At a designated time, the CPE wells also receive 100tissue culture infectious doses (100 TCID50s) of titered influenzavirus, 72 hr later the cell viability is determined. The effectivecompound concentrations which reduce viral-induced CPE by 25% (IC25),50% (IC50), and 90% (IC90) are calculated by regression analysis withsemi-log curve fitting. Cell viability is assessed using CellTiter-Glo(Promega). The toxic concentration of drug that reduces cell numbers by50% and 90% (TC50 and TC90, respectively) are calculated as well.Selectivity (therapeutic) indices (SI=TC/IC) are also calculated.

In Vivo Testing for Inhibition of Influenza Replication

In vivo testing of compounds can be performed, including testing onmammals such as rats or ferrets. Because ferrets (Mustela putorius furo)are naturally susceptible to infection with human influenza A and Bviruses and their disease resembles that of human influenza, theseanimals have been widely used as a model for influenza viruspathogenesis and immunity studies. See Sidwell, R. W. and D. F. Smee,Antiviral Res, 2000, 48(1): p. 1-16: and Colacino, J. M., D.C. DeLong,J. R. Nelson, W. A. Spitzer, J. Tang, F. Victor, and C. Y. Wu,Antimicrob Agents Chemother, 1990, 34(11): p. 2156-63. Ferrets are alsothe model of choice for the study of avian influenza virus H5N1pathogenesis in mammals. See also Zitzow, L. A., T. Rowe, T. Morken,W.-J. Shieh, S. Zaki, and J. M. Katz, Pathogenesis of Avian Influenza A(H5N1) Viruses in Ferrets, 2002. p, 4420-4429. The activities of the PB1Stapled Peptides can be compared to Ribavirin or Oseltamivir as apositive control.

Briefly, young adult male or female ferrets (five ferrets for eachtreatment group) that are serologically negative by hemagglutinationinhibition assay for currently circulating human influenza A or Bviruses are quarantined at least 4 days prior to infection in a BSL-3+animal holding area, where they are housed in cages contained inbioclean portable laminar flow clean room enclosures (Lab Products,Seaford, Del.). Prior to infection, baseline temperatures are measuredtwice daily for at least 3 days. Ferrets are anesthetized with ketamine(25 mg/kg), xylazine (2 mg/kg), and atropine (0.05 mg/kg) by theintramuscular route and infected intranasally (i.n.) with virus/mL inphosphate-buffered saline (PBS) delivered to the nostrils. Controlanimals are mock-infected with an equivalent dilution (1:30) ofnoninfectious allantoic fluid. Stapled Peptides are administered i.v, ori.p. one hour after virus infection. Temperatures are measured twicedaily using either a rectal thermometer or a subcutaneous implantabletemperature transponder (BioMedic Data Systems, Inc. Seaford, Del.) withpre-infection values averaged to obtain a baseline temperature for eachferret. The change in temperature (in degrees Celsius) is calculated ateach time point for each animal. Clinical signs of sneezing (beforeanesthesia), inappetence, dyspnea, and level of activity are assessed. Ascoring system is also used to assess the activity level, and based onthe daily scores for each animal in a group a relative inactivity indexwill be calculated. Rectal temperature and activity scores are used toassess the severity of influenza infection and the ability of StapledPeptides to prevent flu symptoms

Assaying Inhibition of Viral Polymerase Complex Assembly and Activity

The technique of Bimolecular Fluorescence Complementation (“BiFC”) canbe used to assay the compounds. In this technique. N- and C-terminalfragments of fluorescent proteins (e.g. GFP or its derivatives) arefused to interacting proteins. The two non-functional halves of thefluorophore, following the expression in cells, are brought into closeproximity as a result of the specific protein interactions, whichinitiates folding of the fragments into an active protein and results ina detectable fluorescent signal at the site of the protein-proteincomplex. Thus, through BiFC, the specific interaction between PB1 and PAsubunits can be visualized, quantified and localized within live cells.By disrupting PB1-PA interaction with a compound, the BiFC signal willbe reduced, indicative of the presence of potential inhibitors targetingthe assembly of PB1-PA complex. See Hemerka et. al., J. Virol. 2009,3944-3955.

Manufacturing of the Aqueous Pharmaceutical Formulation

In another aspect of the disclosure relates to a method of making theaqueous pharmaceutical formulations disclosed herein. The methodcomprising the steps of dissolving at least one peptidomimeticmacrocycle, or a pharmaceutically acceptable salt thereof in an aqueoussolution. The method can further comprise of stirring the peptidemixture for some additional time. For example, the peptide mixture canbe allowed to be stirred for an additional period of 1 min, 2 min, 3min, 4 min, 5 min, 6 min, 7 min, 8 min, 9 min, 10 min, 15 min, 30 min,45 min, 1 h, 1.5 h, 2 h or more.

In some embodiments, the peptidomimetic macrocycle is added to theaqueous solution at once. In some embodiments, the peptidomimeticmacrocycle is added slowly to the aqueous solution, for example over aperiod of at least about 1 min, 2 min, 3 min, 4 min, 5 min, 6 min, 7min, 8 min, 9 min, 10 min, 15 min, 30 min, 45 min, I h, 1.5 h, 2 h ormore. In some embodiments, the peptidomimetic macrocycle is added slowlyover a period of at most about 2 h, 1.5 h, 1 h, 45 min, 30 min, 15 min,10 min, 9 min, 8 min, 7 min, 6 min, 5 min, 4 min, 3 min, 2 min, 1 min orless.

The aqueous solution comprises an aqueous diluent. The amount of theaqueous diluent can be in the range of about 10-99% w/v of formulation.In some embodiments, the amount of aqueous diluent in the formulation isabout 50-99% w/v. For example, the amount of aqueous diluent is in therange of about 50-95% w-/v, about 50-90% w/v, about 50-85% w/v, about50-80% w/v, about 50-75% w/v, about 50-70% w/v, about 50-65% w/v, about50-60% w/v, about 50-55% w/v, about 55-95% w/v, about 55-90% w/v, about55-85% w/v, about 55-80% w/v, about 55-75% w/v, about 55-70% w/v, about55-65% w/v, about 55-60% w/v, about 60-95% w/v, about 60-90% w/v, about60-85% w/v, about 60-80% w/v, about 60-75% w/v, about 60-70% w/v, about60-65% w/v, about 65-95% w/v, about 65-90% w/v, about 65-85% w/v, about65-80% w/v, about 65-75% w/v, about 65-70% w/v, about 70-95% w/v, about70-90% w/v, about 70-85% w/v, 70-80% w/v, about 70-75% w/v, about 75-95%w/v, about 75-90% w/v, about 75-85% w/v, about 75-80% w/v, about 70-95%w/v, about 70-90% w/v, about 70-85% w/v, about 70-80% w/v, about 70-75%w/v, about 75-95% w/v, about 75-90% w/v, about 75-85% w/v, about 75-80%w/v, about 80-95% w/v, about 80-90% w/v, about 80-85% w/v, about 85-95%w/v, about 85-90% w/v, or about 90-95% w/v. In some embodiments, theamount of aqueous diluent in a formulation is about 85-99% w/v. In someembodiments, the amount of the aqueous diluent in a formulation is about85% w/v, about 86% w/v, about 87% w/v, about 88% w/v, about 89% w/v,about 90% w/v, about 91% w/v, about 92% w/v, about 93% w/v, about 94%w/v, about 95% w/v, about 96% w/v, about 97% w/v, about 98% w/v, orabout 99% w/v. In some embodiments, the amount of the aqueous diluent inthe formulation is about 90% w/v. In some embodiments, the amount of thediluent in the formulation is about 10% w/v, about 20% w/v, about 30%w/v, about 40% w/v, about 50% w/v, about 60% w/v, about 70% w/v, about80% w/v, or about 90% w/v. In some embodiments the diluent is water (forexample, water for injection) and it comprises about 90% w/v of theformulation.

The amount of a peptidomimetic macrocycle in the aqueous pharmaceuticalformulations disclosed herein can range from about 0.0001-50.0% w/v. Forexample, the amount of the a peptidomimetic macrocycle can be about0.0001-10.0 w/v %, about 0.005-10.0% w/v, about 0.01-10.0% w/v, about0.05-10.0% w/v, about 0.1-10.0% w/v, about 0.5-10.0% w/v, about1.0-10.0% w/v, about 2.0-10.0% w/v, about 3.0-10.0% w/v, about 4.0-10.0%w/v, about 5.0-10.0% w/v, 6.0-10.0% w/v, about 7.0-10.0% w/v, about8.0-10.0% w/v, about 9.0-10.0% w/v, about 0.0001-5.0 w/v %, about0.005-5.0% w/v, about 0.01-5.0% w/v, about 0.05-5.0% w/v, about 0.1-5.0%w/v, about 0.5-5.0% w/v, about 1.0-5.0% w/v, about 2.0-5.0% w/v, about3.0-5.0% w/v, about 4.0-5.0% w/v, about 0.0001-2.0 w/v %, about0.005-2.0% w/v, about 0.01-2.0% w/v, about 0.05-2.0% w/v, about 0.1-2.0%w/v, about 0.5-2.0% w/v, or about 1.0-2.0% w/v. In some embodiments, thepeptidomimetic macrocycle is a p53-based peptidomimetic macrocycle andthe amount is about 0.1-5.0% w/v, for example about 1.0% w/v, about 1.5%w/v, or about 2.0% w/v.

In some embodiments, the amount of the peptidomimetic macrocycle is inthe range of about 1-20.0% w/v, 5-20.0% w/v, about 7-20.0% w/v, about10-20.0% w/v, about 12-20.0% w/v, 15-20.0% w/v, 17-20.0% w/v, about5-25.0% w/v, 7-25.0% w/v, 10-25.0% w/v, 12-25.0% w/v, 15-25.0% w/v,17-25.0% w/v, 20-25.0% w/v, or 22-25.0% w/v; 5-35.0% w/v, 7-35.0% w/v,10-35.0% w/v, 12-35.0% w/v, 15-35.0% w/v, 17-35.0% w/v, 20-35.0% w/v,22-35.0% w/v, 25-35.0% w/v, 27-35.0% w/v, 30-35.0% w/v, or 32-35.0% w/v;5-40.0% w/v, 7-40.0% w/v, about 10-40.0% w/v, about 12-40.0% w/v, about1540.0% w/v, about 17-40.0% w/v, about 20-40.0% w/v, 2240.0% w/v,25-40.0% w/v, 2740.0% w/v, 30-40.0% w/v, 33-40.0% w/v, 35-40.0% w/v, or37-40.0% w/v; 5-50.0% w/v, 10-50.0% w/v, 12-50.0% w/v, 15-50.0% w/v,20-50.0% w/v, 22-50.0% w/v, 25-50.0% w/v, 27-50.0% w/v, 30-50.0% w/v,32-50.0% w/v, 35-50.0% w/v, 37-50.0% w/v, 40-50.0% w/v, 42-50.0% w/v,45-50.0% w/v, or 47-50.0% w/v.

In some embodiments, the amount of peptidomimetic macrocycle is about0.5%, about 1.0%, about 1.5%, about 2.0%, about 2.5%, about 3.0%, about3.5%, about 4.0%, about 4.5%, about 5.0%, about 5.5%, about 6.0%, about6.5%, about 7.0%, about 7.5%, about 8.0%, about 8.5%, about 9.0%, about9.5, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%,about 40%, about 45%, or about 50% w/v.

The concentration of a peptidomimetic macrocycle in the aqueouspharmaceutical formulations disclosed herein can be in the range ofabout 1-100 mg/mL. In some embodiments, the amount of a peptidomimeticmacrocycle in the formulation is about 1-5 mg/mL, about 1-10 mg/mL,about 1-15 mg/mL, about 1-20 mg/mL, about 1-25 mg/mL, about 1-30 mg/mL,about 1-35 mg/mL, about 140 mg/mL, about 145 mg/mL, about 1-50 mg/mL,about 1-60 mg/mL, about 1-70 mg/mL, about 1-80 mg/mL, about 1-90 mg/mL,about 5-10 mg/mL, about 5-15 mg/mL, about 5-20 mg/mL, about 5-25 mg/mL,about 5-30 mg/mL, about 5-35 mg/mL, about 540 mg/mL, about 545 mg/mL,about 5-50 mg/mL. about 5-60 mg/mL, about 5-70 mg/mL, about 5-80 mg/mL,about 5-90 mg/mL, about 5-100 mg/mL, about 10-15 mg/mL, about 10-20mg/mL, about 10-25 mg/mL. about 10-30 mg/mL, about 10-35 mg/mL, about10-40 mg/mL, about 10-45 mg/mL, about 10-50 mg/mL, about 10-60 mg/mL,about 10-70 mg/mL, about 10-80 mg/mL, about 10-90 mg/mL, about 10-100mg/mL, about 15-20 mg/mL, about 15-25 mg/mL, about 15-30 mg/mL, about15-35 mg/mL, about 15-40 mg/mL, about 1545 mg/mL, about 15-50 mg/mL,about 15-60 mg/mL, about 15-70 mg/mL, about 15-80 mg/mL, about 15-90mg/mL, about 15-100 mg/mL, about 20-25 mg/mL, about 20-30 mg/mL, about20-35 mg/mL, about 20-40 mg/mL, about 2045 mg/mL, about 20-50 mg/mL,about 20-60 mg/mL, about 20-70 mg/mL, about 20-80 mg/mL, about 20-90mg/mL, about 20-100 mg/mL, about 25-30 mg/mL, about 25-35 mg/mL, about25-40 mg/mL, about 25-45 mg/mL, about 25-50 mg/mL, about 25-60 mg/mL.about 25-70 mg/mL, about 25-80 mg/mL, about 25-90 mg/mL. about 25-100mg/mL, about 30-35 mg/mL, about 30-40 mg/mL, about 3045 mg/mL, about30-50 mg/mL, about 30-60 mg/mL, about 30-70 mg/mL, about 30-80 mg/mL,about 30-90 mg/mL, about 30-100 mg/mL, about 3540 mg/mL, about 35-45mg/mL, about 35-50 mg/mL, about 35-60 mg/mL, about 35-70 mg/mL, about35-80 mg/mL, about 35-90 mg/mL, about 35-100 mg/mL, about 40-45 mg/mL,about 40-50 mg/mL, about 40-60 mg/mL, about 40-70 mg/mL, about 40-80mg/mL, about 40-90 mg/mL, about 45-50 mg/mL, about 45-60 mg/mL, about45-70 mg/mL, about 45-80 mg/mL, about 45-90 mg/mL, about 40-100 mg/mL,about 50-60 mg/mL, about 50-70 mg/mL, about 50-80 mg/mL, about 50-90mg/mL, about 50-100 mg/mL, about 60-70 mg/mL, about 60-80 mg/mL, about60-90 mg/mL, about 60-100 mg/mL, about 70-80 mg/mL, about 70-90 mg/mL,about 70-100 mg/mL, about 80-90 mg/mL, about 80-100 mg/mL or about90-100 mg/mL. In some embodiments, the amount of the peptidomimeticmacrocycles in the formulations of the disclosure can be about 1 mg/mL,about 2 mg/mL, about 3 mg/mL, about 4 mg/mL, about 5 mg/mL, about 6mg/mL, about 7 mg/mL, about 8 mg/mL, about 9 mg/mL, about 10 mg/mL,about 11 mg/mL, about 12 mg/mL, about 13 mg/mL, about 14 mg/mL, about 15mg/mL, about 16 mg/mL, about 17 mg/mL, about 18 mg/mL, about 19 mg/mL,or about 20 mg/mL. In some embodiments, the amount of the peptidomimeticmacrocycles is about 5 mg/mL, about 10 mg/mL, about 15 mg/mL or about 20mg/mL. In some embodiments, the peptidomimetic macrocycle is a p53-basedpeptidomimetic macrocycle and the amount is about 1-20 mg/mL, forexample about 1.0 mg/mL, about 5 mg/mL, about 10 mg/mL, about 15 mg/mLor about 20 mg/mL.

In some embodiments, the aqueous solution further comprises a bufferingagent. In such embodiments, the method of making the aqueouspharmaceutical formulations disclosed herein comprises dissolving atleast one buffering agent in the aqueous diluent, and adding at leastone peptidomimetic macrocycle or a pharmaceutically acceptable saltthereof. In some embodiments, the peptidomimetic macrocycle is added atonce. In some embodiments, the peptidomimetic macrocycle is added slowlyover a period of time as described above. As described above, the methodcan further comprise of stirring the peptide mixture for some additionaltime.

The concentration of the buffering solution can be about 0.01-100 mM. Insome embodiments the concentration of the buffering solution is at least0.1 mM, 1 mM, 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90mM, 100 mM. In some embodiments the concentration of the bufferingsolution is at most 0.1 mM, 1 mM, 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60mM, 70 mM, 80 mM, 90 mM, 100 mM. In some embodiments, the concentrationof the buffering agent is about 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7mM, 8 mM, 9 mM, 10 mM, 11 mM, 12 mM, 13 mM, 14 mM, 15 mM, 16 mM, 17 mM,18 mM, 19 mM, 20 mM, 21 mM, 22 mM, 23 mM, 24 mM, 25 mM, 26 mM, 27 mM, 28mM, 29 mM, 30 mM, 31 mM, 32 mM, 33 mM, 34 mM, 35 mM, 36 mM, 37 mM, 38mM, 39 mM, 40 mM, 41 mM, 42 mM, 43 mM, 44 mM, 45 mM, 46 mM, 47 mM, 48mM, 49 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, or 100 mM.

The method can further involve maintaining the pH of the formulation.For example, maintaining the pH of the reaction medium while thepeptidomimetic macrocycle is being added and/or dissolved therein. ThepH can be maintained by the addition of a pH adjusting agent. Anysuitable pH adjusting agents as described above and throughout thedisclosure can be used.

Non-limiting examples of suitable pH adjusting agents which can beincluded in the methods disclosed herein are hydrochloric acid, sodiumhydroxide, citric acid, phosphoric acid, lactic acid, tartaric acid,succinic acid, or mixtures thereof. In one embodiment, the pH adjustingagent is hydrochloric acid. In one embodiment, the pH adjusting agent issodium hydroxide. In one embodiment, the pH adjusting agent isphosphoric acid. In one embodiment, the pH adjusting agent is lacticacid. In one embodiment, the pH adjusting agent is tartaric acid. In oneembodiment, the pH adjusting agent is tartaric acid. In one embodiment,the pH adjusting agent is succinic acid. In one embodiment, thebuffering agent is a phosphate buffer and the pH adjusting agent insodium hydroxide. For example, the buffering agent can be NaH₂PO₄ andthe pH adjusting agent can be sodium hydroxide, or the buffering agentcan be Na₂HPO₄ and the pH adjusting agent can be sodium hydroxide, otherbuffering agent can be a mixture of NaH₂PO₄ and Na₂HPO₄ and the pHadjusting agent can be sodium hydroxide, or buffering agent can beKH₂PO₄ and the pH adjusting agent can be sodium hydroxide, or thebuffering agent can be K₂HPO₄ and the pH adjusting agent can be sodiumhydroxide, or the buffering agent can be a mixture of KH₂PO₄ and K₂HPO₄and the pH adjusting agent can be sodium hydroxide.

In some embodiments the amount of the pH adjusting agent added to theaqueous pharmaceutical formulation is in the range of about 0.001-1%w/v. For example, in some embodiments, the amount of the pH adjustingagent present is in the range of 0.01-0.1% w/v, 0.1-1% w/v, 0.005-1%w/v, 0.05-1% w/v, 0.5-1% w/v, 0.001-0.5% w/v, 0.01-0.5% w/v, 0.1-0.5%w/v, 0.001-0.1% w/v, or 0.01-0.1 vv. In some embodiments, the amount ofthe pH adjusting agent present in the formulation is in the range ofabout 0.01-0.1% w/v. In some embodiments, the amount of the pH adjustingagent present in the formulation is at least 0.01% w/v, 0.02% w/v, 0.03%w/v, 0.04% w/v, 0.05% w/v, 0.06% w/v, 0.07% w/v, 0.08% w/v, 0.09% w/v,or 0.1% w/v. In some embodiments, the amount of the pH adjusting agentpresent in the formulation is at most 0.1% w/v, 0.09% w/v, 0.08% w/v,0.07% w/v, 0.06% w/v, 0.05% w/v, 0.04% w/v, 0.03% w/v, 0.02% w/v, 0.01%w/v.

In some embodiments the amount of the pH adjusting agent added to theaqueous pharmaceutical formulation is in the range of about 0.01-100mg/mL. For example, in some embodiments, the amount of the pH adjustingagent present is in the range of 0.01-50 mg/mL, 0.01-10 mg/mL, 0.1-100mg/mL, 0.1-50 mg/mL, 0.1-10 mg/mL, 1-100 mg/mL, 1-50 mg/mL, or 1-10mg/mL. In some embodiments, the amount of the pH adjusting agent presentin the formulation is in the range of about 1-10 mg/mL. In someembodiments, the amount of the pH adjusting agent present in theformulation is at least I mg/mL, 2 mg/mL, 3 mg/mL, 4 mg/mL, 5 mg/mL, 6mg/mL, 7 mg/mL, 8 mg/mL, 9 mg/mL, or 10 mg/mL. In some embodiments, theamount of the pH adjusting agent present in the formulation is at most10 mg/mL, 9 mg/mL, 8 mg/mL, 7 mg/mL, 6 mg/mL, 5 mg/mL, 4 mg/mL, 3 mg/mL,2 mg/mL, 1 mg/mL. In some embodiments, the amount of the pH adjustingagent present in the formulation is about 1 mg/mL, about 1.5 mg/mL,about 2 mg/mL, about 2.5 mg/mL, about 3 mg/mL, about 3.5 mg/mL, about 4mg/mL, about 4.5 mg/mL, about 5 mg/mL, about 6 mg/mL, about 7 mg/mL,about 8 mg/mL, about 9 mg/mL, about 10 mg/mL, about 12 mg/mL, about 14mg/mL, about 16 mg/mL, about 18 mg/mL, or about 20 mg/mL. In someembodiments, the amount of the pH adjusting agent present in theformulation is of the pH adjusting agent is present in about 5 mg/mL ofthe formulation.

In some embodiments, the aqueous solution comprises a stabilizing agent.In such embodiments, the method of making the aqueous pharmaceuticalformulations disclosed herein comprises dissolving at least onestabilizing agent in at least an aqueous diluent, and adding at leastone peptidomimetic macrocycle or a pharmaceutically acceptable saltthereof. In some embodiments, the peptidomimetic macrocycle is added atonce. In some embodiments, the peptidomimetic macrocycle is added slowlyover a period of time as described above. As described above, the methodcan further comprise of stirring the peptide mixture for some additionaltime.

In amount of the stabilizing agent in the formulations can be in therange of about 0.001-1% w/v. For example, in the range of about0.001-0.01%, about 0.001-0.1% w/v, about 0.001-0.5% w/v, about 0.01-0.1%w/v, about 0.01-0.5% w/v, about 0.01-0.1% w/v, about 0.1-0.5% w/v orabout 0.5-1% v/v. In some embodiments, the amount of the stabilizingagent in the formulation is about 0.01-0.1% w/v. In some embodiments,the amount of the stabilizing agent is at least about 0.01% w/v, about0.02% w/v, about 0.03% w/v, about 0.04% w/v, about 0.05% w/v, about0.06% w/v, about 0.07% w/v, about 0.08% w/v, about 0.09% w/v, or about0.1% w/v. In some embodiments, the amount of the stabilizing agent is atmost about 0.1% w/v, about 0.09% w/v, about 0.08% w/v, about 0.07% w/v,about 0.06% w/v, about 0.05% w/v, about 0.04% w/v, about 0.03% w/v,about 0.02% w/v, about 0.01% w/v. In some embodiments, the amount of thestabilizing agent in the formulation is about 0.01% w/v, about 0.02%w/v, about 0.03% w/v, about 0.04% w/v, about 0.05% w/v, about 0.06% w/v,about 0.07% w/v, about 0.08% w/v, about 0.09% w/v, or about 0.1% w/v. Insome embodiments, the amount of the stabilizing agent in the formulationis about 0.01% w/v. In some embodiments, the amount of the stabilizingagent in the formulation is about 0.02% w/v. In some embodiments, theamount of the stabilizing agent in the formulation is about 0.03% w/v.In some embodiments, the amount of the stabilizing agent in theformulation is about 0.04% w/v. In some embodiments, the amount of thestabilizing agent in the formulation is about 0.05% w/v.

In some embodiments the amount of the stabilizing agent is about 0.01-10mg/mL. For example, in some embodiments, the amount of the stabilizingagent is about 0.01-5 mg/mL, about 0.01-1 mg/mL, about 0.01-0.5 mg/mL,about 0.01-0.1 mg/mL, about 0.1-10 mg/mL, about 0.1-5 mg/mL, about 0.1-1mg/mL, about 0.1-0.5 mg/mL, about 1-10 mg/mL, or about 1-5 mg/mL. Insome embodiments, the amount of the stabilizing agent in the formulationis in the range of about 0.01-1.0 mg/mL.

In some embodiments, the amount of the stabilizing agent is at leastabout 0.1 mg/mL, about 0.2 mg/mL, about 0.3 mg/mL, about 0.4 mg/mL,about 0.5 mg/mL, about 0.6 mg/mL, about 0.7 mg/mL, about 0.8% mg/mL,about 0.9 mg/mL, or about 1 mg/mL. In some embodiments, the amount ofthe stabilizing agent is at most about 1 mg/mL, about 0.9 mg/mL, about0.8 mg/mL, about 0.7 mg/mL, about 0, 6 mg/mL, about 0.5 mg/mL, about 0.4mg/mL, about 0.3 mg/mL, about 0.2 mg/mL, or about 0.1 mg/mL.

In some embodiments, the amount of the stabilizing agent is about 0.1mg/mL, about 0, 2 mg/mL, about 0.3 mg/mL, about 0.4 mg/mL, about 0.5mg/mL, about 0.6 mg/mL, about 0.7 mg/mL, about 0.8 mg/mL, about 0.9mg/mL, or about 1 mg/mL. In some embodiments, the amount of thestabilizing agent in the formulation is about 0.1 mg/mL. In someembodiments, the amount of the stabilizing agent in the formulation isabout 0.2 mg/mL. In some embodiments, the amount of the stabilizingagent in the formulation is about 0.3 mg/mL. In some embodiments, theamount of the stabilizing agent in the formulation is about 0.4 mg/mL.In some embodiments, the amount of the stabilizing agent in theformulation is about 0.5 mg/mL.

In some embodiments, the aqueous solution comprises both a bufferingagent and stabilizing agent. In such embodiments, the method of makingthe aqueous pharmaceutical formulations disclosed herein comprisesdissolving at least one stabilizing agent and at least one bufferingagent in an aqueous diluent, and adding at least one peptidomimeticmacrocycle or a pharmaceutically acceptable salt thereof. In someembodiments, the peptidomimetic macrocycle is added at once. In someembodiments, the peptidomimetic macrocycle is added slowly over a periodof time as described above. As described above, the method can furthercomprise of stirring the peptide mixture for some additional time.

In some examples, the method of making the aqueous pharmaceuticalformulations disclosed herein comprises dissolving at least onebuffering agent, at least one tonicity adjusting agent and at least onestabilizing agent in at least one aqueous diluent, and adding at leastone peptidomimetic macrocycle or a pharmaceutically acceptable saltthereof. In some embodiments, the buffering agent, the tonicityadjusting agent and the stabilizing agents are dissolved in the aqueousdiluent in this order. In some embodiments, the peptidomimeticmacrocycle is added at once. In some embodiments, the peptidomimeticmacrocycle is added slowly over a period of time as described above.

The amount of the tonicity adjusting agent in the aqueous pharmaceuticalformulations disclosed herein can be in the range of about 0.001-50%w/v, for example about 0.001-0.1% w/v, about 0.001-1.0% w/v, about0.001-10% w/v, about 1-10% w/v, about 1-20% w/v, about 1-30% w/v, about1-40% w/v, about 1-50% w/v, about 5-10% w/v, about 5-20% w/v, about5-30% w/v, about 5-40% w/v, about 5-50% w/v, about 10-20% w/v, about10-30% w/v, about 10-40% w/v, about 10-50% w/v, about 15-20% w/v, about15-30% w/v, about 15-40% w/v, about 15-50% w/v, about 20-30% w/v, about20-40% w/v, about 20-50% w/v, about 25-30% w/v, about 25-40% w/v, about25-50% w/v, about 30-40% w/v, about 30-50,% w/v, about 35-40% w/v, about35-50% w/v, about 40-50% w/v, or about 45-50% w/v. In some embodiments,the amount of the tonicity adjusting agent is about 1% w/v, about 2%w/v, about 3% w/v, about 4% w/v, about 5% w/v, about 6% w/v, about 7%w/v, about 8% w/v, about 9% w/v, or about 10% w/v. In some embodiments,the amount of the tonicity adjusting agent is about 7% w/v. In someembodiments. the amount of the tonicity adjusting agent is about 8% w/v.In some embodiments, the amount of the tonicity adjusting agent is about9% w/v. In some embodiments, the amount of the tonicity adjusting agentis about 10% w/v.

The concentration of the tonicity adjusting agent can vary in the rangeof about 1-500 mg/mL. For example, the concentration of the tonicityadjusting agent in the aqueous pharmaceutical formulations disclosedherein can be in the range of about 1-400 mg/mL, 1-300 mg/mL, 1-200mg/mL, 1-100 mg/mL, 10-500 mg/mL, 10-400 mg/mL, 10-300 mg/mL, 10-200mg/mL, 10-100 mg/mL, 20-500 mg/mL, 20-400 mg/mL, 20-300 mg/mL, 20-200mg/mL, 20-100 mg/mL, 30-500 mg/mL, 30-400 mg/mL, 30-300 mg/mL, 30-200mg/mL, 30-100 mg/mL, 40-500 mg/mL, 40-400 mg/mL, 40-300 mg/mL, 40-200mg/mL, 40-100, mg, 50-500 mg/mL, 50-400 mg/mL, 50-300 mg/mL, 50-200mg/mL, 50-100 mg/mL, 60-500 mg/mL, 60-400 mg/mL, 60-30 mg/mL, 60-200mg/mL, 60-100 mg/mL, 70-500 mg/mL, 70400 mg/mL, 70-300 mg/mL, 70-200mg/mL, 70-100 mg/mL, 80-500 mg/mL, 80400 mg/mL, 80-300 mg/mL, 80-200mg/mL, 80-200 mg/mL, 90-500 mg/mL, 90-400 mg/mL, 90-300 mg/mL, 90-200mg/mL, 90-100 mg/mL, 100-500 mg/mL, 100400 mg/mL, 100-300 mg/mL, 100-200mg/mL, 200-500 mg/mL, 200-400 mg/mL, 200-300 mg/mL, 300-500 mg/mL,300400 mg/mL or 400-500 mg/mL. In some embodiments, the concentration ofthe tonicity adjusting agent is about 10 mg/mL, about 20 mg/mL, about 30mg/mL, about 40 mg/mL, about 50 mg/mL, about 60 mg/mL, about 70 mg/mL,about 80 mg/mL, about 90 mg/mL, or about 100 mg/mL. In some embodiments,the concentration of the tonicity adjusting agent is about 50 mg/mL. Insome embodiments, the concentration of the tonicity adjusting agent isabout 80 mg/mL. In some embodiments, the concentration of the tonicityadjusting agent is about 100 mg/mL. In some embodiments, the tonicityadjusting agent is trehalose (for example, D- trehalose) and theconcentration is about 80 mg/mL.

In some embodiments, the concentration of the tonicity adjusting agentis between about 100-500 mM. For example the concentration of thetonicity adjusting agent in the aqueous pharmaceutical formulationsdisclosed herein can be 100-400 mM, 100-300 mM, 100-200 mM, 200-500 mM,200400 mM, 200-300 mM, 300-500 mM, 300-400 mM or 400-500 mM. In someembodiments, the concentration of the tonicity adjusting agent isbetween about 200-300 mM, for example 210-300 mM, 220-300 mM, 230-300mM, 240-300 mM, 250-300 mM, 260-300 mM, 270-300 mM, 280-300 mM, 290-300mM, 200-290 mM, 210-290 mM, 220-290 mM, 230-290 mM, 240-290 mM, 250-290mM, 260-290 mM, 270-290 mM, 280-290 mM, 200-280 mM, 210-280 mM, 220-280mM, 230-280 mM, 240-280 mM, 250-280 mM, 260-280 mM, 270-280 mM, 200-270mM, 210-270 mM, 220-270 mM, 230-270 mM, 240-270 mM, 250-270 mM, 260-270mM, 200-260 mM, 210-260 mM, 220-260 mM, 230-260 mM, 240-260 mM, 250-260mM, 200-250 mM, 210-250 mM, 220-250 mM, 230-250 mM, 240-250 mM, 200-240mM, 210-240 mM, 220-240 mM, 230-240 mM, 200-230 mM, 210-230 mM, 220-230mM, 200-220 mM, 210-220 mM, or 210-220 mM. In some embodiments, theconcentration of the tonicity adjusting agent is between about 220-260mM. For example, about 220 mM, 230 mM, 240 mM, 250 mM, or 260 mM.

The methods described herein can additionally comprise addition of oneor more optional excipients and/or ingredients. For example addition ofone or more antioxidants, antimicrobial agent, surfactants, lubricants,thickening agents. preservatives, chelating agents.

In some embodiments the amount of antioxidants used is in the range ofabout 0.001-5% w/v, for example about 0.001-4.5%, 0.0014%, 0.001-3%,0.001-2%, 0.002-1%, 0.001-0.5%, or 0.001-0.05% w/v. In some embodimentsthe amount of antioxidants used is in the range of about 0.001-about0.5%, about 0.1-about 0.5%, about 0.2-about 0.5%, about 0.3-about 0.5%,about 0.4-about 0.5%, about 0.01-about 0.4%, about 0.1-about 0.4%, about0.2-about 0.4%, about 0.3-about 0.4%, about 0.01-about 0.3%, about0.1-about 0.3%, about 0.2-about 0.3%, about 0.01-about 0.2%, about0.1-about 0.2%, or about 0.01-about 0.1% w/v.

Such antimicrobial agents can be employed at a level of from about0.005-0.5% w/v, for example about 0.001-0.01% w/v, about 0.01-0.1% w/v,about 0.1-0.5% w/v or about 0.01-0.05% w/v.

The methods described herein can additionally comprise prefilteringand/or clarifying the peptidomimetic formulation by a suitable process,for example by centrifugation or by filtration. Filtration can be by anysuitable means, for example by depth filter media or by membranefilters. In some embodiments, filtration can be by means of a 0.22micrometer filters.

The method can optionally involve sterilization of the aqueouspharmaceutical formulations. Sterilization can be performed by anysuitable technique. For example, a suitable sterilization method caninclude one or more of sterile filtration, chemical, irradiation heatfiltration, and addition of a chemical disinfectant to the aqueouspharmaceutical formulation. In some examples, the formulations aresterilized by moist heat sterilization. In some examples, theformulations are sterilized by dry heat sterilization. In some examples,the formulations are sterilized by chemical cold sterilization. In someexamples, the formulations are sterilized by radiation sterilization. Insome examples, the formulations are sterilized by filtration. In someexamples, the formulations are sterilized by filtration using anappropriate micron sterilizing grade filters. The filtration can becarried out by any suitable means, e.g. cellulose-based filters,cellulosic esters (MCE), polyvinylidene fluoride (PVDF),polytetrafluoroethylene (PTFE), or polyethersulfone (PES) filters. Insome embodiments PVDF filters are used. Filters of any appropriatemicron size can be used. In some embodiments, the filter size can b^(e)0.001-0.5 micrometer, for example 0.001-0.01 micrometer, 0.01-0.1micrometer, 0.1-0.2 micrometer, 0.2-0.3 micrometer, 0.3-0.4 3 micrometeror 0.4-0.5 micrometer. In some embodiments 0.22 micrometer filters areused. In some embodiments 0.22 micrometer PVDF filters are used.

The aqueous pharmaceutical formulations can be in a form that issuitable for direct administration or can be in a concentrated form thatrequires dilution relative to what is administered to the patient. Forexample, aqueous pharmaceutical formulations, described in thisdisclosure, can be in a form that is suitable for direct administrationwithout any further dilution or reconstitution. The formulations can bediluted or reconstituted prior to administration with a suitable aqueousdiluent(s) to obtain a finished concentration. The diluent can be aninjection or infusion fluid. Examples of injection or infusion fluidinclude, but are not limited to, WFI (Bacteriostatic Water ForInjection), SWFI (Sterile Water For Injection), D5W (Dextrose 5% inWater), DlOW (Dextrose 10% in Water), D5LR (Dextrose in Lactate Ringer'sSolution), D5 ¼ S (Dextrose 5% in ¼ Strength Saline (5% Dextrose and0.22% Sodium Chloride Injection)), D5 ½ S (Dextrose 5% in ½ StrengthSaline (5% Dextrose and 0.45% Sodium Chloride Injection)), D5NS(Dextrose 5% in Normal Saline (5% Dextrose and 0.9% Sodium ChlorideInjection)), D5R (Dextrose 5% in Ringer's Injection), DIONS (Dextrose10% in Normal Saline (10% Dextrose and 0.9% Sodium Chloride Injection)),ISIOW (Invert Sugar 10% in Saline (10% Invert Sugar in 0.9% SodiumChloride Injection)), LR (Lactated Ringer's Injection), Pr (ProteinHydrolysate Injection), R (Ringer's Injection), NS Sodium Chloride 0.9%(Normal Saline), SOD CL 5 (Sodium Chloride 5% (5% Sodium ChlorideInjection), and Sod Lac (Sodium Lactate, ⅙ Molar (M/6 Sodium LactateInjection)). In some examples, the formulations can be diluted with 0.9%sodium chloride, 5% dextrose in water (D5W), 5% dextrose in normalsaline (D5NS), 5 dextrose in half amount of normal saline (D5 ½NS),lactated ringer's injection or a mixture thereof.Dilution/reconstitution can be performed immediately prior to theadministration. In some cases, dilution/reconstitution can be performedshortly before the administration. In some cases, the dilution isperformed at most 1 min, 5 min, 15 min, 30 min, 45 min, 60 min, 90 min,2 h, 3 h, 4 h, 5 h, 6 h, 7 h, 8 h, 9 h, 10 h, 11 h, 12 h, 13 h, 14 h, 15h, 16 h, 17 h, 18 h, 19 h, 20 h, 21 h, 22 h, 23 h, or 24 h before theadministration to the subject. In some examples the reconstituted anddiluted solutions is used within 1-10 hours, 2-8 hours, 3-7 hours, 4-6hours reconstitution and/or dilution. In some examples, the formulationsare diluted/reconstituted more than 1 day, 2 days, 3 days, 4 days, 5days, 6 days, 1 week before administration.

FIG. 1 depicts the manufacturing process of an exemplary aqueousformulation according to the disclosure.

Purity, Stability and Degradation

The formulations of the disclosure can be characterized by low endotoxinconcentration. In some embodiments, the formulations can have aconcentration of endotoxin of less than about 100 EU/mL, for example,less than about 90 EU/mL, 80 EU/mL, 70 EU/mL, 60 EU/mL, 50 EU/mL, 40EU/mL, 30 EU/mL, 20 EU/mL, 10 EU/mL, 5 EU/mL, 1 EU/mL, 0.5 EU/mL, 0.2EU/mL, 0.1 EU/mL, 0.05 EU/mL, 0.01 EU/mL, 0.005 EU/mL, or 0.001 EU/mL.In some embodiments the concentration of the endotoxin is 0.1-10 EU/mL,for example about 0.1-1 EU/mL, 0.1-2 EU/mL, 0.1-3 EU/mL, 0.1-4 EU/mL,0.1-5 EU/mL, 0.1-6 EU/mL, 0.1-7 EU/mL, 0.1-8 EU/mL, 0.1-9 EU/mL, 1-2EU/mL, 1-3 EU/mL, 1-4 EU/mL, 1-5 EU/mL, 1-6, 1-7, 1-8, 1-9, 1-10, 2-3EU/mL, 2-4 EU/mL, 2-5 EU/mL, 2-6, 2-7, 2-8, 2-9, 2-10, 3-4 EU/mL, 3-5EU/mL, 3-6 EU/mL, 3-7 EU/mL, 3-8 EU/mL, 3-9 EU/mL, 3-10 EU/mL, 4-5EU/mL, 4-6 EU/mL, 4-7 EU/mL, 4-8 EU/mL, 4-9 EU/mL, 4-10 EU/mL, 5-6EU/mL, 5-7 EU/mL, 5-8 EU/mL, 5-9 EU/mL, 5-10 EU/mL, 6-7 EU/mL, 6-8EU/mL, 6-9 EU/mL, 6-10 EU/mL, 7-8 EU/mL, 7-9 EU/mL, 7-10 EU/mL, 8-9EU/mL, 8-10 EU/mL, or 9-10 EU/mL.

In some embodiments the formulations of the disclosure are essentiallyparticulate-free solutions. In some embodiments, the formulation isessentially free of particles of size greater than about 1 μm, 2 μm, 3μm, 4 μm, 5 μm, 6 μm, 7 μm, 8 μm, 9 μm, 10 μm, 11 μm, 12 μm, 13 μm, 14μm, 15 μm, 16 μm, 17 μm, 18 μm, 19 μm, 20 μm, 21 μm, 22 μm, 23 μm, 24μm, 25 μm, 26 μm, 27 μm, 28 μm, 29 μm, 30 μm, 35 μm, 40 μm, 45 μm, 50μm, or more.

In some embodiments, the formulation comprise at most about 1, about 2,about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10,about 11, about 12, about 13, about 14, about 15, about 16, about 17,about 18, about 19, about 20, about 21, about 22, about 23, about 24,about 25, about 26. about 27, about 28, about 29, about 30, about 50,about 100, about 200, about 300, about 400, about 500, about 600, about700, about 800, about 900, about 1,000, about 1,100, about 1,200, about1,300, about 1,400, about 1,500, about 1,600, about 1,700, about 1,800,about 1,900, about 2,000, about 2,200, about 2,400, about 2,600, about2,800, about 3,000, about 3,500, about 4,000, about 4,500, about 5,000,about 5,500, about 6,000, about 6,500, about 7,000, about 8,000, about8,500, about 9,000, about 9,500, or about 10,000 particles of sizegreater than or equal to 10 μm per 1 mL or 5 mL of formulation. In someembodiments the formulations of the disclosure are essentially free ofparticles of size greater than or equal to 10 μm. In some embodimentsthe formulations of the disclosure less than 500 particles of sizegreater than or equal to 10 μm in per I mL or 5 mL of formulation. Insome embodiments the formulations of the disclosure less than 1000particles of size greater than or equal to 10 μm in per 1 mL or 5 mL offormulation. In some embodiments the formulations of the disclosure lessthan 1200 particles of size greater than or equal to 10 μm in per 1 mLor 5 mL of formulation. In some embodiments the formulations of thedisclosure less than 1,000-1,200 particles of size greater than or equalto 10 μm in per 1 mL or 5 mL of formulation.

In some embodiments. the formulation comprise at most about 1, about 2,about 3, about 4. about 5. about 6, about 7, about 8, about 9, about 10,about 11, about 12, about 13, about 14, about 15, about 16, about 17,about 18, about 19, about 20, about 21, about 22, about 23, about 24,about 25, about 26, about 27, about 28, about 29, about 30, about 50,about 60, about 70, about 80, about 90, about 100, about 110, about 120,about 130, about 140, about 150, about 200, about 300, about 400, about500, about 600, about 700, about 800, about 900, about 1,000, about1,100, about 1,200, about 1,300, about 1,400, about 1,500, about 1,600,about 1,700, about 1,800, about 1,900, about 2,000, about 2,500, about3,000, about 3,500, about 4,000, about 4,500, about 5,000, about 5,500,or about 6,000 particles of size greater than or equal to 25 μm per I mLor 5 mL of formulation. In some embodiments the formulations of thedisclosure are essentially free of particles of size greater than orequal to 25 μm. In some embodiments the formulations comprise at most 50particles of size greater than or equal to 25 μm per 1 mL or 5 mL offormulation. In some embodiments the formulations comprise at most 100particles of size greater than or equal to 25 μm per 1 mL or 5 mL offormulation. In some embodiments the formulations comprise at most 120particles of size greater than or equal to 25 μm in per 1 mL or 5 mL offormulation. In some embodiments the formulations comprise about 100-120particles of size greater than or equal to 25 μm per 1 mL or 5 mL offormulation.

In some embodiments the formulations of the disclosure are essentiallyfree of particles of size greater than or equal to 50 μm. In someembodiments the formulations comprise at most I particles of sizegreater than or equal to 50 μm per 1 mL or 5 mL of formulation. In someembodiments the formulations comprise at most 2 particles of sizegreater than or equal to 50 μm per 1 mL or 5 mL of formulation. In someembodiments the formulations comprise at most 3 particles of sizegreater than or equal to 50 μm in per 1 mL or 5 mL of formulation. Insome embodiments the formulations comprise about 1-5 particles of sizegreater than or equal to 50 μm per 1 mL or 5 mL of formulation. In someembodiments the formulations of the disclosure are essentially free ofparticles of size greater than or equal to 50 μm. In some embodimentsthe formulations comprise at most 1 particles of size greater than orequal to 50 μm per container. In some embodiments the formulationscomprise at most 2 particles of size greater than or equal to 50 μm percontainer. In some embodiments the formulations comprise at most 3particles of size greater than or equal to 50 μm in per container. Insome embodiments the formulations comprise about 1-5 particles of sizegreater than or equal to 25 μm container.

In some embodiments, the formulations comprise 0-10000, 100-10,000,500-10,000, 1,000-10,000, 1,500-10,000, 2,000-10,000, 2,500-10,000,3,000-10,000, 3,500-10,000, 4,000-10,000, 4,500-10,000, 5,000-10.000,5,500-10,000, 6,000-10,000, 6,500-10,000, 7,000-10,000, 7,500-10,000,8,000-10,000, 8,500-10,000, 9,000-10,000, or 9,500-10,000 particles ofsize greater than or equal to 10 μm per mL of formulation.

In some embodiments, the formulations comprise 0-10000, 100-10,000,500-10,000, 1.000-10,000, 1,500-10,000, 2,000-10,000, 2,500-10,000,3,000-10,000, 3,500-10,000, 4,000-10,000, 4,500-10,000, 5,000-10,000,5,500-10,000, 6,000-10,000, 6,500-10,000, 7,000-10,000, 7,500-10,000,8,000-10,000, 8,500-10,000, 9,000-10,000, or 9,500-10,000 particles ofsize greater than or equal to 10 μm per 5 mL of formulation.

In some embodiments, the formulations comprise 0-10000, 100-10,000,500-10,000, 1,000-10,000, 1.500-10,000, 2,000-10,000, 2,500-10,000,3,000-10,000, 3,500-10,000, 4,000-10,000, 4,500-10,000, 5,000-10,000,5,500-10,000, 6,000-10,000, 6,500-10,000, 7,000-10,000, 7,500-10,000,8,000-10,000, 8,500-10,000, 9,000-10,000, or 9,500-10,000 particles ofsize greater than or equal to 25 μm per mL of formulation.

In some embodiments, the formulations comprise 0-10000, 100-10,000,500-10,000, 1.000-10,000, 1,500-10,000, 2,000-10,000, 2,500-10,000,3,000-10,000, 3,500-10,000, 4,000-10,000, 4,500-10,000, 5,000-10,000,5,500-10,000, 6,000-10,000, 6,500-10,000, 7,000-10,000, 7,500-10,000,8,000-10,000, 8,500-10,000, 9,000-10,000, or 9,500-10,000 particles ofsize greater than or equal to 25 μm per 5 mL of formulation.

In some embodiments, the formulations comprise 0-10000, 100-10,000,500-10,000, 1,000-10,000, 1.500-10,000, 2,000-10,000, 2,500-10,000,3,000-10,000, 3,500-10,000, 4,000-10,000, 4,500-10,000, 5,000-10,000,5,500-10,000, 6,000-10,000, 6,500-10,000, 7,000-10,000, 7,500-10,000,8,000-10,000, 8,500-10,000, 9,000-10,000, or 9,500-10,000 particles ofsize greater than or equal to 50 μm per 1 mL of formulation

In some embodiments, the formulations comprise 0-10000, 100-10,000,500-10,000, 1.000-10,000, 1,500-10,000, 2,000-10,000, 2.500-10,000,3,000-10,000, 3,500-10,000, 4,000-10.000, 4,500-10,000, 5,000-10,000,5,500-10,000, 6,000-10,000, 6,500-10,000, 7,000-10,000, 7,500-10,000,8,000-10,000, 8,500-10,000, 9,000-10,000, or 9,500-10,000 particles ofsize greater than or equal to 50 μm per 5 mL of formulation.

In some embodiments, the formulations of the present disclosure canremain stable after exposure to a single or multiple freeze-thaw events.Formulations of the present disclosure can also remain stable afterexposure to physical agitation, such as one would expect to encounterupon shipping product from one location to another. Stability can bemeasured by any one of a number of different ways, including visualinspection for precipitate formation, analysis of percent peptidomimeticmacrocycle remaining in solution after exposure to stress conditions(e.g., by size-exclusion HPLC), or analysis of the formation of chemicalvariants and/or decomposition products of the peptidomimetic macrocycle(e.g., by anion exchange or reverse phase HPLC analysis). In someembodiments of the present disclosure, no precipitate visible to thenaked eye is formed in the formulation after at least one freeze thawevent. In some embodiments the formulation remains stable after at leastthree freeze thaw events. In some embodiments the formulation remainsstable after at least six freeze thaw events. In some embodiments, atleast 80, 85, 90%, 95%, 96%, 975, 98%, or 99% of the peptidomimeticmacrocycle remains in the formulation after at least one freeze thawevent.

In some embodiments, the total peptidomimetic degradation productsformed in the formulations of the present disclosure is less than 1.0%when stored at a temperature of 40° C. for a period of one month. Insome further embodiments, the total degradation products of the compoundof Formula 1 formed is less than about 0.9%, about 0.8%, about 0.7%,about 0.6%, about 0.5%, about 0.4%, about 0.3%, about 0.2%, or about0.1% when stored at a temperature of 40° C. for a period of one month.

In some embodiments, the total peptidomimetic degradation productsformed in the formulations of the present disclosure is less than 1.0%when stored at a temperature of 40° C. for a period of about two months,about three months, about four months, about five months about sixmonths.

In some further embodiments, the amount of any single impurity in theformulation at any storage temperature is less than 0.001%, 0.01%, 0.1%,0.5%, 1.0%, 1.5%, 2.0%, 2.5%, 3.0%, 3.5%, 4.0%, 4.5% 0.0%, 5.5%, 6.0%,6.5%, 7.0%, 7.5%, 8.0%, 8.5%, 9.0%, 9,5%, or 10%. In some furtherembodiments, the amount of any single impurity in the formulation at anystorage temperature is less than 1.0%. In some further embodiments. theamount of any single impurity in the formulation at any storagetemperature is less than 2.0%. In some further embodiments, the amountof any single impurity in the formulation at any storage temperature isless than 3.0%.

In some further embodiments, the amount of any single impurity in theformulation at any storage temperature is less than 4.0%. In somefurther embodiments, the amount of any single impurity in theformulation at any storage temperature is less than 5.0%.

In some further embodiments, the amount of any single impurity in theformulation at any storage temperature is less than 0.5% when stored ata temperature of −20° C. for a period of 0 months, 0.5 months, 1.0months, 1.5 months, 2.0 months, 2.5 months, 3.0 months, 3.5 months, 4.0months, 4.5 months, 5.0 months, 5.5 months, 6.0 months, 8 months, 10months, 12 months, or more months. In some further embodiments, theamount of any single impurity in the formulation at any storagetemperature is less than 1% when stored at a temperature of −20° C. fora period of 0 months, 0.5 months, 1.0 months, 1.5 months, 2.0 months,2.5 months, 3.0 months, 3.5 months, 4.0 months, 4.5 months, 5.0 months,5.5 months, 6.0 months, 8 months, 10 months, 12 months, or more months.In some further embodiments. the amount of any single impurity in theformulation at any storage temperature is less than 1.5% when stored ata temperature of −20° C. for a period of 0 months, 0.5 months, 1.0months, 1.5 months, 2.0 months, 2.5 months, 3.0 months, 3.5 months, 4.0months, 4.5 months, 5.0 months, 5.5 months, 6.0 months, 8 months, 10months, 12 months, or more months. In some further embodiments, theamount of any single impurity in the formulation at any storagetemperature is less than 2.0% when stored at a temperature of −20° C.for a period of 0 months, 0.5 months, 1.0 months, 1.5 months, 2.0months, 2.5 months, 3.0 months, 3.5 months, 4.0 months, 4.5 months, 5.0months, 5.5 months, 6.0 months, 8 months, 10 months, 12 months, or moremonths. In some further embodiments, the amount of any single impurityin the formulation at any storage temperature is less than 2.5% whenstored at a temperature of −20° C. for a period of 0 months, 0.5 months,1.0 months, 1.5 months, 2.0 months, 2.5 months, 3.0 months, 3.5 months,4.0 months, 4.5 months, 5.0 months, 5.5 months, 6.0 months, 8 months, 10months, 12 months, or more months. In some further embodiments, theamount of any single impurity in the formulation at any storagetemperature is less than 3.0% when stored at a temperature of −20° C.for a period of 0 months, 0.5 months, 1.0 months, 1.5 months, 2.0months, 2.5 months, 3.0 months, 3.5 months, 4.0 months, 4.5 months, 5.0months, 5.5 months, 6.0 months, 8 months, 10 months, 12 months, or moremonths. In some further embodiments, the amount of any single impurityin the formulation at any storage temperature is less than 5.0% whenstored at a temperature of −20° C. for a period of 0 months, 0.5 months,1.0 months, 1.5 months, 2.0 months, 2.5 months, 3.0 months, 3.5 months,4.0 months, 4.5 months, 5.0 months, 5.5 months, 6.0 months, 8 months, 10months, 12 months, or more months.

In some further embodiments. the amount of any single impurity in theformulation at any storage temperature is less than 0.5% when stored ata temperature of 5° C. for a period of 0 months, 0.5 months, 1.0 months,1.5 months, 2.0 months, 2.5 months, 3.0 months, 3.5 months, 4.0 months,4.5 months, 5.0 months, 5.5 months, 6.0 months, 8 months, 10 months, 12months, or more months. In some further embodiments, the amount of anysingle impurity in the formulation at any storage temperature is lessthan 1% when stored at a temperature of 5° C. for a period of 0 months,0.5 months, 1.0 months, 1.5 months, 2.0 months, 2.5 months, 3.0 months,3.5 months, 4.0 months, 4.5 months, 5.0 months, 5.5 months, 6.0 months,8 months, 10 months, 12 months, or more months. In some furtherembodiments, the amount of any single impurity in the formulation at anystorage temperature is less than 1.5% when stored at a temperature of 5°C. for a period of 0 months, 0.5 months, 1.0 months, 1.5 months, 2.0months, 2.5 months, 3.0 months, 3.5 months, 4.0 months, 4.5 months, 5.0months, 5.5 months, 6.0 months, 8 months, 10 months, 12 months, or moremonths. In some further embodiments, the amount of any single impurityin the formulation at any storage temperature is less than 2.0% whenstored at a temperature of 5° C. for a period of 0 months, 0.5 months,1.0 months, 1.5 months, 2.0 months, 2.5 months, 3.0 months, 3.5 months,4.0 months, 4.5 months, 5.0 months, 5.5 months, 6.0 months, 8 months, 10months, 12 months, or more months. In some further embodiments, theamount of any single impurity in the formulation at any storagetemperature is less than 2.5% when stored at a temperature of 5° C. fora period of 0 months, 0.5 months, 1.0 months, 1.5 months, 2.0 months,2.5 months, 3.0 months, 3.5 months, 4.0 months, 4.5 months, 5.0 months,5.5 months, 6.0 months, 8 months, 10 months, 12 months, or more months.In some further embodiments, the amount of any single impurity in theformulation at any storage temperature is less than 3.0% when stored ata temperature of 5° C. for a period of 0 months, 0.5 months, 1.0 months,1.5 months, 2.0 months, 2.5 months, 3.0 months, 3.5 months, 4.0 months,4.5 months, 5.0 months, 5.5 months, 6.0 months, 8 months, 10 months, 12months, or more months. In some further embodiments, the amount of anysingle impurity in the formulation at any storage temperature is lessthan 5.0% when stored at a temperature of 5° C. for a period of 0months, 0.5 months, 1.0 months, 1.5 months, 2.0 months, 2.5 months, 3.0months, 3.5 months, 4.0 months, 4.5 months, 5.0 months, 5.5 months, 6.0months, 8 months, 10 months, 12 months, or more months.

In some further embodiments, the amount of any single impurity in theformulation at any storage temperature is less than 0.5% when stored ata temperature of 25° C. for a period of 0 months, 0.5 months, 1.0months, 1.5 months, 2.0 months, 2.5 months, 3.0 months, 3.5 months, 4.0months, 4.5 months, 5.0 months, 5.5 months, 6.0 months, 8 months, 10months, 12 months, or more months. In some further embodiments, theamount of any single impurity in the formulation at any storagetemperature is less than 1% when stored at a temperature of 25° C. for aperiod of 0 months, 0.5 months, 1.0 months, 1.5 months, 2.0 months, 2.5months, 3.0 months, 3.5 months, 4.0 months, 4.5 months, 5.0 months, 5.5months, 6.0 months, 8 months, 10 months, 12 months, or more months. Insome further embodiments, the amount of any single impurity in theformulation at any storage temperature is less than 1.5% when stored ata temperature of 25° C. for a period of 0 months, 0.5 months, 1.0months, 1.5 months, 2.0 months, 2.5 months, 3.0 months, 3.5 months, 4.0months, 4.5 months, 5.0 months, 5.5 months, 6.0 months, 8 months, 10months, 12 months, or more months. In some further embodiments, theamount of any single impurity in the formulation at any storagetemperature is less than 2.0% when stored at a temperature of 25° C. fora period of 0 months, 0.5 months, 1.0 months, 1.5 months, 2.0 months,2.5 months, 3.0 months, 3.5 months, 4.0 months, 4.5 months, 5.0 months,5.5 months, 6.0 months, 8 months, 10 months, 12 months, or more months.In some further embodiments, the amount of any single impurity in theformulation at any storage temperature is less than 2.5% when stored ata temperature of 25° C. for a period of 0 months, 0.5 months, 1.0months, 1.5 months, 2.0 months, 2.5 months, 3.0 months, 3.5 months, 4.0months, 4.5 months, 5.0 months, 5.5 months, 6.0 months, 8 months, 10months, 12 months, or more months. In some further embodiments, theamount of any single impurity in the formulation at any storagetemperature is less than 3.0% when stored at a temperature of 25° C. fora period of 0 months, 0.5 months, 1.0 months, 1.5 months, 2.0 months,2.5 months, 3.0 months, 3.5 months, 4.0 months, 4.5 months, 5.0 months,5.5 months, 6.0 months, 8 months, 10 months, 12 months, or more months.In some further embodiments, the amount of any single impurity in theformulation at any storage temperature is less than 5.0% when stored ata temperature of 25° C. for a period of 0 months, 0.5 months, 1.0months, 1.5 months, 2.0 months, 2.5 months, 3.0 months, 3.5 months, 4.0months, 4.5 months, 5.0 months, 5.5 months, 6.0 months, 8 months, 10months, 12 months, or more months.

In some further embodiments, the amount of any single impurity in theformulation at any storage temperature is less than 0.5% when stored ata temperature of 40° C. for a period of 0 months, 0.5 months, 1.0months, 1.5 months, 2.0 months, 2.5 months, 3.0 months, 3.5 months, 4.0months, 4.5 months, 5.0 months, 5.5 months, 6.0 months, 8 months, 10months, 12 months, or more months. In some further embodiments, theamount of any single impurity in the formulation at any storagetemperature is less than 1% when stored at a temperature of 40° C. for aperiod of 0 months, 0.5 months, 1.0 months, 1.5 months, 2.0 months, 2.5months, 3.0 months, 3.5 months, 4.0 months, 4.5 months, 5.0 months, 5.5months, 6.0 months, 8 months, 10 months, 12 months, or more months. Insome further embodiments, the amount of any single impurity in theformulation at any storage temperature is less than 1.5% when stored ata temperature of 40° C. for a period of 0 months, 0.5 months, 1.0months, 1.5 months, 2.0 months, 2.5 months, 3.0 months, 3.5 months, 4.0months, 4.5 months, 5.0 months, 5.5 months, 6.0 months, 8 months, 10months, 12 months, or more months. In some further embodiments, theamount of any single impurity in the formulation at any storagetemperature is less than 2.0% when stored at a temperature of 40° C. fora period of 0 months, 0.5 months, 1.0 months, 1.5 months, 2.0 months,2.5 months, 3.0 months, 3.5 months, 4.0 months, 4.5 months, 5.0 months,5.5 months, 6.0 months, 8 months, 10 months, 12 months, or more months.In some further embodiments, the amount of any single impurity in theformulation at any storage temperature is less than 2.5% when stored ata temperature of 40° C. for a period of 0 months, 0.5 months, 1.0months, 1.5 months, 2.0 months, 2.5 months, 3.0 months, 3.5 months, 4.0months, 4.5 months, 5.0 months, 5.5 months, 6.0 months, 8 months, 10months, 12 months, or more months. In some further embodiments, theamount of any single impurity in the formulation at any storagetemperature is less than 3.0% when stored at a temperature of 40° C. fora period of 0 months, 0.5 months, 1.0 months, 1.5 months, 2.0 months,2.5 months, 3.0 months, 3.5 months, 4.0 months, 4.5 months, 5.0 months,5.5 months, 6.0 months, 8 months, 10 months, 12 months, or more months.In some further embodiments, the amount of any single impurity in theformulation at any storage temperature is less than 5.0% when stored ata temperature of 40° C. for a period of 0 months, 0.5 months, 1.0months, 1.5 months, 2.0 months, 2.5 months, 3.0 months, 3.5 months, 4.0months, 4.5 months, 5.0 months, 5.5 months, 6.0 months, 8 months, 10months, 12 months, or more months.

In some cases the pharmaceutically acceptable formulation expires inabout 1-5 years. In some cases the formulation expires in about 1, 2, 3or 4 years. In some cases the formulation expires in more than 5 years.In some cases the formulation expires in less than a year. In some casesthe formulation expires in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 months.

In some cases the total amount of peptidomimetic degradation products atthe time of product expiration are in the range of above 0.1-10%. Insome cases the total degradation product at the time of expiration is inthe range of about 0.01-1, about 0.01-2, about 0.01-3, about 0.01-4,about 0.01-5, about 0.01-6, about 0.01-7, about 0.01-8, or about 0.01-9.about 1-2, about 1-3, about 1-4, about 1-5, about 1-6, about 1-7. about1-8, about 1-9, about 2-3, about 3-4, about 2-5, about 2-6, about 2-7,about 2-8, about 2-9, about 3-4, about 3-5, about 3-6, about 3-7, about3-8, about 3-9, about 3-10, about 4-5, about 4-6, about 4-7, about 4-8,about 4-9, about 4-10, about 5-6, about 5-7, about 5-8, about 5-9, about5-10, about 6-7, about 6-8, about 6-9, about 6-10, about 7-8, about 7-9,about 7-10, about 8-9, about 8-10 or about 9-10%. In some embodimentsthe amount of total degradation product at the time of expiration isabout 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%,about 8%. about 9%, about 10%. In some embodiments the amount of totaldegradation product at the time of expiration is about 0.01%, about0.05%, about 0.1%, about 0.15%, about 0.2%, about 0.25%, about 0.3%,about 0.35%, about 0.40%, about 0.45%, about 0.50%, about 0.55%, about0.60%, about 0.65%, about 0.70%, about 0.75%, about 0.80%, about 0.85%,about 0.90%, about 0.95%, or about 1.0%.

In some cases aqueous pharmaceutical formulations of the instantdisclosure are stored at −40 to 65° C., for example from −5 to 40° C. Insome cases the formulations can be stored at about −40° C., about −30°C., −20° C., −10° C., −5° C., 0° C., about 5° C., about 10° C., about15° C., about 20° C., about 25° C., about 30° C., about 35° C., about40° C., about 45° C., about 50° C., about 55° C., about 60° C., or about65° C. In some embodiments, the formulations are stored at or belowambient temperature. In some embodiments, the formulations are storedabove ambient temperature.

Sparging

In some embodiments the stability of the peptidomimetic macrocycles inthe formulations of the disclosure can be improved by sparging theformulation with an inert gas. A variety of inert gases can be used as asparging material including but not limited to nitrogen, argon, helium,or a combination thereof. In some embodiments the inert gas is nitrogen.The sparging is generally carried out till the oxygen is reduced orcompletely removed from the formulations peptidomimetic macrocycles. Thetime period for sparging depends in several factors including the amountof formulation, the effectiveness of agitation and the flow rate of theinert gas. In some embodiments, sparging is done by bubbling the inertgas through the formulations for a period of about 1 min-12 h. In someembodiments the formulations are sparged for a period of about 1min-about 11 h, about 1 min-about 10 h, about 1 min-9 h, about 1 min-8h, about 1 min-7 h, about 1 min-6 h, about 1 min-5 h, about 1 min-4 h,about 1 min-3 h, about 1 min-2 h, about 1 min-1 h, about 1 min-45 min,about 1 min-about 30 min, about 1 min-15 min, about 1 min-10 min, about1 min-about 9 min, about 1 min-8 min, about 1 min-about 7 min, about 1min-6 min, about 1 min-about 5 min, about 1 min-about 4 min, about 1min-about 3 min, about 1 min-about 2 min. In some embodiments, spargingis performed for less than about 1 minute.

Methods of Use Methods

In one aspect, provided herein are aqueous pharmaceutical formulationsthat are useful in competitive binding assays to identify agents whichbind to the natural ligand(s) of the proteins or peptides upon which thepeptidomimetic macrocycles are modeled. For example, in the p53/MDMXsystem, labeled peptidomimetic macrocycles based on p53 can be used in aMDMX binding assay along with small molecules that competitively bind toMDMX. Competitive binding studies allow for rapid in vitro evaluationand determination of drug candidates specific for the p53/MDMX system.Such binding studies can be performed with any of the peptidomimeticmacrocycles disclosed herein and their binding partners.

Further provided are methods for the generation of antibodies againstthe peptidomimetic macrocycles. In some embodiments, these antibodiesspecifically bind both the peptidomimetic macrocycle and the precursorpeptides, such as p53, to which the peptidomimetic macrocycles arerelated. Such antibodies, for example, disrupt the nativeprotein-protein interaction, for example, binding between p53 and MDMX.

In other aspects, provided herein are both prophylactic and therapeuticmethods of treating a subject at risk of (or susceptible to) a disorderor having a disorder associated with aberrant (e.g., insufficient orexcessive) expression or activity of the molecules including p53, MDM2or MDMX.

In another embodiment, a disorder is caused, at least in part, by anabnormal level of p53 or MDM2 or MDMX, (e.g., over or under expression),or by the presence of p53 or MDM2 or MDMX exhibiting abnormal activity.As such, the reduction in the level and/or activity of p53 or MDM2 orMDMX, or the enhancement of the level and/or activity of p53 or MDM2 orMDMX, by peptidomimetic macrocycles derived from p53, is used, forexample, to ameliorate or reduce the adverse symptoms of the disorder.

In another aspect, provided herein are methods for treating orpreventing a disease including hyperproliferative disease andinflammatory disorder by interfering with the interaction or bindingbetween binding partners, for example, between p53 and MDM2 or p53 andMDMX. These methods comprise administering an effective amount of acompound to a warm blooded animal, including a human. In someembodiments, the administration of one or more compounds disclosedherein induces cell growth arrest or apoptosis.As used herein, the term “treatment” is defined as the application oradministration of a therapeutic agent to a patient, or application oradministration of a therapeutic agent to an isolated tissue or cell linefrom a patient, who has a disease, a symptom of disease or apredisposition toward a disease, with the purpose to cure, heal,alleviate, relieve, alter, remedy, ameliorate, improve or affect thedisease, the symptoms of disease or the predisposition toward disease.

Disease and Disorders

In some embodiments, the pharmaceutical formulations can be used totreat, prevent, and/or diagnose cancers and neoplastic conditions. Asused herein, the terms “cancer”. “hyperproliferative” and “neoplastic”refer to cells having the capacity for autonomous growth, i.e., anabnormal state or condition characterized by rapidly proliferating cellgrowth. Hyperproliferative and neoplastic disease states can becategorized as pathologic, i.e., characterizing or constituting adisease state, or can be categorized as non-pathologic, i.e., adeviation from normal but not associated with a disease state. The termis meant to include all types of cancerous growths or oncogenicprocesses, metastatic tissues or malignantly transformed cells, tissues,or organs, irrespective of histopathology type or stage of invasiveness.A metastatic tumor can arise from a multitude of primary tumor types,including but not limited to those of breast, lung, liver, colon andovarian origin. “Pathologic hyperproliferative” cells occur in diseasestates characterized by malignant tumor growth. Examples ofnon-pathologic hyperproliferative cells include proliferation of cellsassociated with wound repair. Examples of cellular proliferative and/ordifferentiative disorders include cancer, e.g., carcinoma, sarcoma, ormetastatic disorders. In some embodiments, the pharmaceuticalformulations can be used for controlling/treating breast cancer, ovariancancer, colon cancer, lung cancer, metastasis of such cancers and thelike.

Examples of cancers or neoplastic conditions include, but are notlimited to, a fibrosarcoma, myosarcoma. liposarcoma, chondrosarcoma,osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma,lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma,Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, gastric cancer,esophageal cancer, rectal cancer, pancreatic cancer, ovarian cancer,prostate cancer, uterine cancer, cancer of the head and neck, skincancer, brain cancer, squamous cell carcinoma, sebaceous glandcarcinoma, papillary carcinoma, papillary adenocarcinoma,cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renalcell carcinoma. hepatoma, bile duct carcinoma, choriocarcinoma,seminoma, embryonal carcinoma, Wilm's tumor, cervical cancer, testicularcancer, small cell lung carcinoma, non-small cell lung carcinoma,bladder carcinoma, epithelial carcinoma, glioma, astrocytoma,medulloblastoma, craniopharyngioma, ependymoma, pinealoma,hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma,melanoma, neuroblastoma, retinoblastoma, leukemia, lymphoma, Kaposisarcoma, or glioblastoma multiforme.

In some embodiments, the cancer is head and neck cancer, melanoma, lungcancer, breast cancer, or glioma.

In some examples, the cancer is pancreatic cancer, bladder cancer, coloncancer, liver cancer, colorectal cancer (colon cancer or rectal cancer),breast cancer, prostate cancer, renal cancer, hepatocellular cancer,lung cancer, ovarian cancer, cervical cancer, gastric cancer, esophagealcancer, head and neck cancer, melanoma. neuroendocrine cancers, CNScancers, brain tumors, bone cancer, skin cancer, ocular tumor,choriocarcinoma (tumor of the placenta), sarcoma or soft tissue cancer.

In some examples, cancer is bladder cancer, bone cancer, breast cancer,cervical cancer, CNS cancer, colon cancer, ocular tumor, renal cancer,liver cancer, lung cancer, pancreatic cancer, choriocarcinoma (tumor ofthe placenta), prostate cancer, sarcoma, skin cancer, soft tissue canceror gastric cancer.

In some examples, the cancer is breast cancer. Non limiting examples ofbreast cancer that can be treated by the instant pharmaceuticalformulations include ductal carcinoma in situ (DCIS or intraductalcarcinoma), lobular carcinoma in situ (LCIS), invasive (or infiltrating)ductal carcinoma, invasive (or infiltrating) lobular carcinoma.inflammatory breast cancer, triple-negative breast cancer, paget diseaseof the nipple, phyllodes tumor (phylloides tumor or cystosarcomaphyllodes), angiosarcoma, adenoid cystic (or adenocystic) carcinoma,low-grade adenosquamous carcinoma, medullary carcinoma, papillarycarcinoma, tubular carcinoma, metaplastic carcinoma, micropapillarycarcinoma, and mixed carcinoma.

In some examples, the cancer is bone cancer. Non limiting examples ofbone cancer that can be treated by the instant pharmaceuticalformulations include osteosarcoma, chondrosarcoma, the Ewing SarcomaFamily of Tumors (ESFTs).

In some examples, the cancer is skin cancer. Non limiting examples ofskin cancer that can be treated by the instant pharmaceuticalformulations include melanoma, basal cell skin cancer, and squamous cellskin cancer.

In some examples, the cancer is ocular tumor. Non limiting examples ofocular tumor that can be treated by the pharmaceutical formulations ofthe instant disclosure include ocular tumor is choroidal nevus,choroidal melanoma, choroidal metastasis, choroidal hemangioma,choroidal osteoma, iris melanoma, uveal melanoma, melanocytoma,metastasis retinal capillary hemangiomas, congenital hypertrophy of theRPE, RPE adenoma or retinoblastoma.

Examples of proliferative disorders include hematopoietic neoplasticdisorders. As used herein, the term “hematopoietic neoplastic disorders”includes diseases involving hyperplastic/neoplastic cells ofhematopoietic origin, e.g., arising from myeloid, lymphoid or erythroidlineages, or precursor cells thereof. The diseases can arise from poorlydifferentiated acute leukemias, e.g., crythroblastic leukemia and acutemegakaryoblastic leukemia. Additional exemplary myeloid disordersinclude, but are not limited to, acute promyeloid leukemia (APML), acutemyelogenous leukemia (AML) and chronic myelogenous leukemia (CML)(reviewed in Vaickus (1991), Crit Rev. Oncol. Hemotol. 11:267-97);lymphoid malignancies include, but are not limited to acutelymphoblastic leukemia (ALL) which includes B-lineage ALL and T-lineageALL, chronic lymphocytic leukemia (CLL), prolymphocytic leukemia (PLL),hairy cell leukemia (HLL) and Waldenstrom's macroglobulinemia (WM).Additional forms of malignant lymphomas include, but are not limited tonon-Hodgkin lymphoma and variants thereof, peripheral T cell lymphomas,adult T cell leukemia/lymphoma (ATL), cutaneous T-cell lymphoma (CTCL),large granular lymphocytic leukemia (LGF), Hodgkin's disease andReed-Stemberg disease.

Examples of cellular proliferative and/or differentiative disorders ofthe breast include, but are not limited to, proliferative breast diseaseincluding, e.g., epithelial hyperplasia, sclerosing adenosis, and smallduct papillomas; tumors, e.g., stromal tumors such as fibroadenoma,phyllodes tumor, and sarcomas, and epithelial tumors such as large ductpapilloma: carcinoma of the breast including in situ (noninvasive)carcinoma that includes ductal carcinoma in situ (including Paget'sdisease) and lobular carcinoma in situ, and invasive (infiltrating)carcinoma including, but not limited to, invasive ductal carcinoma,invasive lobular carcinoma, medullary carcinoma, colloid (mucinous)carcinoma, tubular carcinoma, and invasive papillary carcinoma, andmiscellaneous malignant neoplasms. Disorders in the male breast include,but are not limited to, gynecomastia and carcinoma.

Examples of cellular proliferative and/or differentiative disorders ofthe skin include, but are not limited to proliferative skin disease suchas melanomas. including mucosal melanoma, superficial spreadingmelanoma, nodular melanoma, lentigo (e.g. lentigo maligna, lentigomaligna melanoma, or acral lentiginous melanoma), amelanotic melanoma,desmoplastic melanoma, melanoma with features of a Spitz nevus, melanomawith small nevus-like cells, polypoid melanoma, and soft-tissuemelanoma: basal cell carcinomas including micronodular basal cellcarcinoma, superficial basal cell carcinoma, nodular basal cellcarcinoma (rodent ulcer), cystic basal cell carcinoma, cicatricial basalcell carcinoma, pigmented basal cell carcinoma, aberrant basal cellcarcinoma, infiltrative basal cell carcinoma, nevoid basal cellcarcinoma syndrome, polypoid basal cell carcinoma, pore-like basal cellcarcinoma, and fibroepithelioma of Pinkus: squamus cell carcinomasincluding acanthoma (large cell acanthoma), adenoid squamous cellcarcinoma, basaloid squamous cell carcinoma, clear cell squamous cellcarcinoma, signet-ring cell squamous cell carcinoma, spindle cellsquamous cell carcinoma, Marjolin's ulcer, erythroplasia of Queyrat, andBowen's disease; or other skin or subcutaneous tumors.

Examples of cellular proliferative and/or differentiative disorders ofthe lung include, but are not limited to, bronchogenic carcinoma,including paraneoplastic syndromes, bronchioloalveolar carcinoma,neuroendocrine tumors, such as bronchial carcinoid, miscellaneoustumors, and metastatic tumors; pathologies of the pleura, includinginflammatory pleural effusions, noninflammatory pleural effusions,pneumothorax, and pleural tumors, including solitary fibrous tumors(pleural fibroma) and malignant mesothelioma.

Examples of cellular proliferative and/or differentiative disorders ofthe colon include, but are not limited to, non-neoplastic polyps,adenomas, familial syndromes, colorectal carcinogenesis, colorectalcarcinoma, and carcinoid tumors.

Examples of cellular proliferative and/or differentiative disorders ofthe liver include, but are not limited to, nodular hyperplasias,adenomas, and malignant tumors, including primary carcinoma of the liverand metastatic tumors. Examples of cellular proliferative and/ordifferentiative disorders of the ovary include, but are not limited to,ovarian tumors such as, tumors of coelomic epithelium, serous tumors,mucinous tumors, endometrioid tumors, clear cell adenocarcinoma,cystadenofibroma, Brenner tumor, surface epithelial tumors; germ celltumors such as mature (benign) teratomas, monodermal teratomas, immaturemalignant teratomas, dysgerminoma, endodermal sinus tumor,choriocarcinoma: sex cord-stomal tumors such as, granulosa-theca celltumors, thecomafibromas, androblastomas, hill cell tumors, andgonadoblastoma; and metastatic tumors such as Krukenberg tumors.

In one aspect, the present invention provides novel peptidomimeticmacrocycles that are useful in competitive binding assays to identifyagents which bind to the natural ligand(s) of the proteins or peptidesupon which the peptidomimetic macrocycles are modeled. For example, inthe BH3/BCL-X_(L) anti-apoptotic system labeled peptidomimeticmacrocycles based on BH3 can be used in a BCL-X_(L) binding assay alongwith small molecules that competitively bind to BCL-X_(L). Competitivebinding studies allow for rapid in vitro evaluation and determination ofdrug candidates specific for the BH3/BCL-X_(L) system. The inventionfurther provides for the generation of antibodies against thepeptidomimetic macrocycles. In some embodiments, these antibodiesspecifically bind both the peptidomimetic macrocycle and the BH3peptidomimetic precursors upon which the peptidomimetic macrocycles arederived. Such antibodies, for example, disrupt the BH3/BCL-XL systems,respectively.

In other aspects, the present invention provides for both prophylacticand therapeutic methods of treating a subject at risk of (or susceptibleto) a disorder or having a disorder associated with aberrant (e.g.,insufficient or excessive) BCL-2 family member expression or activity(e.g., extrinsic or intrinsic apoptotic pathway abnormalities). It isbelieved that some BCL-2 type disorders are caused, at least in part, byan abnormal level of one or more BCL-2 family members (e.g., over orunder expression), or by the presence of one or more BCL-2 familymembers exhibiting abnormal activity. As such, the reduction in thelevel and/or activity of the BCL-2 family member or the enhancement ofthe level and/or activity of the BCL-2 family member, is used, forexample, to ameliorate or reduce the adverse symptoms of the disorder.

In one embodiment, the compounds of the invention are used to treatdisorders associated with expression or overexpression of Mcl-1. Mcl-1has been shown to be expressed in many tissues and neoplastic cell linesand is thought to participate in the development of malignancies(hallinger et al. (2004) Clin. Cancer Res. 10:4185-4191). Thepeptidomimetic macrocycles of the invention can be used for thetreatment of such malignancies.

In one embodiment, the disorder being treated (e.g. cancer) isdifferentially responsive to the peptidomimetic macrocycles of theinvention. In some embodiments, the cancer is treated with a BIMpeptidomimetic macrocycle and is at least 2-fold less sensitive totreatment using a BID polypeptide (such as a BID peptidomimeticmacrocycle or uncrosslinked polypeptide) as measured in an in vitro cellviability assay. In other embodiments, the cancer is at least 5-foldless sensitive to treatment using a BID polypeptide as measured in an invitro cell viability assay. In yet other embodiments, the cancer is atleast 8-fold less sensitive to treatment using a BID polypeptide asmeasured in an in vitro cell viability assay. In other embodiments, thecancer is treated with a BID peptidomimetic macrocycle and is at least2-fold less sensitive to treatment using a BIM polypeptide (such as aBIM peptidomimetic macrocycle or uncrosslinked polypeptide) as measuredin an in vitro cell viability assay. In other embodiments, the cancer isat least 5-fold less sensitive to treatment using a BIM polypeptide asmeasured in an in vitro cell viability assay. In yet other embodiments,the cancer is at least 8-fold less sensitive to treatment using a BIMpolypeptide as measured in an in vitro cell viability assay.

In another embodiment, a method of treating a human patient is providedcomprising performing an assay to evaluate the levels of a BCL-familyprotein and administering to the patient a peptidomimetic macrocycle ifan aberrant or irregular level of expression of the BCL-family proteinis detected. BCL-family proteins include, for example, BCL-2, BCL-X_(L),MCL-1, Bf11/A1, BOO/DIVA, NRH/NR13, BAX, BAD, BAK, BOK, BIK, PUMA, BIM,BMF, BLK, BNIP3, HRK, NIX, SPIKE, and Noxa. In one embodiment, a methodof treating a human patient is provided comprising performing an assayto evaluate the levels of BCL-2 in the patient and administering to thepatient a peptidomimetic macrocycle if an aberrant or irregular level ofexpression of BCL-2 is detected. In another embodiment, a method oftreating a human patient is provided comprising performing an assay toevaluate the levels of BCL-X_(L) in the patient and administering to thepatient a peptidomimetic macrocycle if an aberrant or irregular level ofexpression of BCL-X_(L) is detected. In another embodiment, a method oftreating a human patient is provided comprising performing an assay toevaluate the levels of MCL-1 in the patient and administering to thepatient a peptidomimetic macrocycle if an aberrant or irregular level ofexpression of MCL-1 is detected. In another embodiment, a method oftreating a human patient is provided comprising performing an assay toevaluate the levels of BAX in the patient and administering to thepatient a peptidomimetic macrocycle if an aberrant or irregular level ofexpression of BAX is detected. In another embodiment, a method oftreating a human patient is provided comprising performing an assay toevaluate the levels of BAD in the patient and administering to thepatient a peptidomimetic macrocycle if an aberrant or irregular level ofexpression of BAD is detected. In another embodiment, a method oftreating a human patient is provided comprising performing an assay toevaluate the levels of BAK in the patient and administering to thepatient a peptidomimetic macrocycle if an aberrant or irregular level ofexpression of BAK is detected. In another embodiment, a method oftreating a human patient is provided comprising performing an assay toevaluate the levels of PUMA in the patient and administering to thepatient a peptidomimetic macrocycle if an aberrant or irregular level ofexpression of PUMA is detected. In another embodiment, a method oftreating a human patient is provided comprising performing an assay toevaluate the levels of Noxa in the patient and administering to thepatient a peptidomimetic macrocycle if an aberrant or irregular level ofexpression of Noxa is detected. In another embodiment, a method oftreating a human patient is provided comprising performing an assay toevaluate the levels of Noxa in the patient and administering to thepatient a peptidomimetic macrocycle if an aberrant or irregular level ofexpression of Noxa is detected. In another embodiment, a method oftreating a human patient is provided comprising performing an assay toevaluate the levels of Bf11/A1 in the patient and administering to thepatient a peptidomimetic macrocycle if an aberrant or irregular level ofexpression of Bf11/A 1 is detected. In another embodiment, a method oftreating a human patient is provided comprising performing an assay toevaluate the levels of BOO/DIVA in the patient and administering to thepatient a peptidomimetic macrocycle if an aberrant or irregular level ofexpression of BOO/DIVA is detected. In another embodiment, a method oftreating a human patient is provided comprising performing an assay toevaluate the levels of NRH/NR 13 in the patient and administering to thepatient a peptidomimetic macrocycle if an aberrant or irregular level ofexpression of NRH/NR13 is detected. In another embodiment, a method oftreating a human patient is provided comprising performing an assay toevaluate the levels of BOK in the patient and administering to thepatient a peptidomimetic macrocycle if an aberrant or irregular level ofexpression of BOK is detected. In another embodiment, a method oftreating a human patient is provided comprising performing an assay toevaluate the levels of BIK in the patient and administering to thepatient a peptidomimetic macrocycle if an aberrant or irregular level ofexpression of BIK is detected. In another embodiment, a method oftreating a human patient is provided comprising performing an assay toevaluate the levels of BMF in the patient and administering to thepatient a peptidomimetic macrocycle if an aberrant or irregular level ofexpression of BMF is detected. In another embodiment, a method oftreating a human patient is provided comprising performing an assay toevaluate the levels of BLK in the patient and administering to thepatient a peptidomimetic macrocycle if an aberrant or irregular level ofexpression of BLK is detected. In another embodiment, a method oftreating a human patient is provided comprising performing an assay toevaluate the levels of BNIP3 in the patient and administering to thepatient a peptidomimetic macrocycle if an aberrant or irregular level ofexpression of BNIP3 is detected. In another embodiment, a method oftreating a human patient is provided comprising performing an assay toevaluate the levels of HRK in the patient and administering to thepatient a peptidomimetic macrocycle if an aberrant or irregular level ofexpression of HRK is detected. In another embodiment, a method oftreating a human patient is provided comprising performing an assay toevaluate the levels of Nix in the patient and administering to thepatient a peptidomimetic macrocycle if an aberrant or irregular level ofexpression of Nix is detected. In another embodiment, a method oftreating a human patient is provided comprising performing an assay toevaluate the levels of SPIKE in the patient and administering to thepatient a peptidomimetic macrocycle if an aberrant or irregular level ofexpression of SPIKE is detected.

In one aspect, the invention provides methods of treating breast cancerby administering the peptidomimetic macrocycles of the invention. Breastcancer includes invasive breast carcinomas, such as invasive ductalcarcinoma, invasive lobular carcinoma, tubular carcinoma, invasivecribriform carcinoma, medullary carcinoma, mucinous carcinoma and othertumours with abundant mucin, cystadenocarcinoma, columnar cell mucinouscarcinoma, signet ring cell carcinoma, neuroendocrine tumours (includingsolid neuroendocrine carcinoma. atypical carcinoid tumour, smallcell/oat cell carcinoma, or large cell neuroendocrine carcioma).invasive papillary carcinoma, invasive micropapillary carcinoma,apocrine carcinoma, metaplastic carcinomas. pure epithelial metaplasticcarciomas, mixed epithelial/mesenchymal metaplastic carcinomas,lipid-rich carcinoma, secretory carcinoma, oncocytic carcinoma, adenoidcystic carcinoma, acinic cell carcinoma, glycogen-rich clear cellcarcinoma, sebaceous carcinoma, inflammatory carcinoma or bilateralbreast carcinoma; mesenchymal tumors such as haemangioma, angiomatosis,haemangiopericytoma, pseudoangiomatous stromal hyperplasia,myofibroblastoma, fibromatosis (aggressive), inflammatorymyofibroblastic tumour, lipoma. angiolipoma, granular cell tumour,neurofibroma, schwannoma, angiosarcoma, liposarcoma, rhabdomyosarcoma.osteosarcoma, leiomyoma, or leiomysarcoma; myoepithelial lesions such asmyoepitheliosis, adenomyoepithelial adenosis, adenomyoepithelioma, ormalignant myoepithelioma; fibroepithelial tumours such as fibroadenoma,phyllodes tumour, low grade periductal stromal sarcoma, or mammaryhamartoma; and tumours of the nipple such as nipple adenoma,syringomatous adenoma, or Paget's disease of the nipple.

Treatment of breast cancer can be effected in conjunction with anyadditional therapy, such as a therapy that is part of the standard ofcare. A surgical technique such as lumpectomy or mastectomy can beperformed prior to, during, or following treatment with thepeptidomimetic macrocycles of the invention. Alternatively, radiationtherapy can be used for the treatment of breast cancer in conjunctionwith the peptidomimetic macrocycles of the invention. In other cases,the peptidomimetic macrocycles of the invention are administered incombination with a second therapeutic agent. Such an agent can be achemotherapeutic agent such as an individual drug or combination ofdrugs and therapies. For example, the chemotherapeutic agent can be anadjuvant chemotherapeutic treatment such as CMF (cyclophosphamide,methotrexate, and 5-fluorouracil); FAC or CAF (5-fluorouracil,doxorubicin, cyclophosphamide); AC or CA (doxorubicin andcyclophosphamide); AC-Taxol (AC followed by paclitaxel); TAC (docetaxel,doxorubicin, and cyclophosphamide): FEC (5-fluorouracil, epirubicin andcyclophosphamide); FECD (FEC followed by docetaxel); TC (docetaxel andcyclophosphamide). In addition to chemotherapy, trastuzumab can also beadded to the regimen depending on the tumor characteristics (i.e.HER2/neu status) and risk of relapse. Hormonal therapy can also beappropriate before, during or following chemotherapeutic treatment. Forexample, tamoxifen can be administered or a compound in the category ofaromatase inhibitors including, but not limited to aminogluthetimide.anastrozole. exemestane, formestane. letrozole, or vorozole. In otherembodiments, an antiangiogenic agent can be used in combination therapyfor the treatment of breast cancer. The antiangiogenic agent can be ananti-VEGF agent including, but not limited to bevacizumab.

In another aspect, the peptidomimetic macrocycles of the invention canbe used to treat ovarian cancer. Ovarian cancers include ovarian tumorssuch as, tumors of coelomic epithelium, serous tumors, mucinous tumors,endomctrioid tumors, clear cell adenocarcinoma, cystadenofibroma.Brenner tumor, surface epithelial tumors; germ cell tumors such asmature (benign) teratomas, monodermal teratomas, immature malignantteratomas, dysgerminoma, endodermal sinus tumor, choriocarcinoma: sexcord-stomal tumors such as, granulosa-theca cell tumors,thecomafibromas, androblastomas, hill cell tumors, and gonadoblastoma;and metastatic tumors such as Krukenbcrg tumors.

The peptidomimetic macrocycles of the invention can be administered inconjunction with a second therapy such as a therapy that is part of thestandard of care. Surgery, immunotherapy, chemotherapy, hormone therapy,radiation therapy, or a combination thereof, are some possibletreatments available for ovarian cancer. Some possible surgicalprocedures include debulking, and a unilateral or bilateral oophorectomyand/or a unilateral or bilateral salpigectomy.

Anti-cancer drugs that can be used include cyclophosphamide, etoposide,altretamine, and ifosfamide. Hormone therapy with the drug tamoxifen canbe used to shrink ovarian tumors. Radiation therapy can be external beamradiation therapy and/or brachytherapy.

In another aspect, the peptidomimetic macrocycles of the invention canbe used to treat prostate cancer. Prostate cancers includeadenocarcinomas and metastasized adenocarcinomas. The peptidomimeticmacrocycles of the invention can be administered in conjunction with asecond therapy such as a therapy that is part of the standard of care.Treatment for prostate cancer can involve surgery, radiation therapy,High Intensity Focused Ultrasound (HIFU), chemotherapy, cryosurgery,hormonal therapy, or any combination thereof. Surgery can involveprostatectomy, radical perineal prostatectomy, laparoscopic radicalprostatectomy, transurethral resection of the prostate or orchiectomy.Radiation therapy can include external beam radiation therapy and/orbrachytherapy. Hormonal therapy can include orchiectomy; administrationof antiandrogens such as flutamide, bicalutamide, nilutamide, orcyproterone acetate: medications which inhibit the production of adrenalandrogens such as DHEA, such as ketoconazole and aminoglutethimide; andGnRH antagonists or agonists such as Abarelix (Plenaxis@), Cetrorelix(Cetrotide@), Ganirelix (Antagont), leuprolide, goserelin, triptorelin,or buserelin. Treatment with an anti-androgen agent, which blocksandrogen activity in the body, is another available therapy. Such agentsinclude flutamide, bicalutamide, and nilutamide. This therapy istypically combined with LHRH analog administration or an orchiectomy,which is termed a combined androgen blockade (CAB). Chemotherapyincludes, but is not limited to, administration of docetaxel, forexample with a corticosteroid such as prednisone. Anti-cancer drugs suchas doxorubicin, estramustine, etoposide, mitoxantrone, vinblastine,paclitaxel, carboplatin can also be administered to slow the growth ofprostate cancer, reduce symptoms and improve the quality of life.Additional compounds such as bisphosphonate drugs can also beadministered.

In another aspect, the peptidomimetic macrocycles of the invention canbe used to treat renal cancer. Renal cancers include, but are notlimited to, renal cell carcinomas, metastases from extra-renal primaryneoplasms, renal lymphomas, squamous cell carcinomas, juxtaglomerulartumors (reninomas), transitional cell carcinomas, angiomyolipomas,oncocytomas and Wilm's tumors. The peptidomimetic macrocycles of theinvention can be administered in conjunction with a second therapy suchas a therapy that is part of the standard of care. Treatment for renalcancer can involve surgery, percutaneous therapies, radiation therapies,chemotherapy, vaccines, or other medication. Surgical techniques usefulfor treatment of renal cancer in combination with the peptidomimeticmacrocycles of the invention include nephrectomy, which can includeremoval of the adrenal gland, retroperitoneal lymph nodes, and any othersurrounding tissues affected by the invasion of the tumor. Percutaneoustherapies include, for example, image-guided therapies which can involveimaging of a tumor followed by its targeted destruction byradiofrequency ablation or cryotherapy. In some cases, otherchemotherapeutic or other medications useful in treating renal cancercan be a-interferon, interleukin-2, bevacizumab, sorafenib, sunitib,temsirolimus or other kinase inhibitors.

In other aspects, the invention provides methods of treating pancreaticcancer by administering peptidomimetic macrocycles of the invention,such as a pancreatic cancer selected from the following: an epitheliodcarcinoma in the pancreatic duct tissue and an adenocarcinoma in apancreatic duct. The most common type of pancreatic cancer is anadenocarcinoma, which occurs in the lining of the pancreatic duct.Possible treatments available for pancreatic cancer include surgery,immunotherapy, radiation therapy, and chemotherapy. Possible surgicaltreatment options include a distal or total pancreatectomy and apancreaticoduodenectomy (Whipple procedure). Radiation therapy can be anoption for pancreatic cancer patients, specifically extemal beamradiation where radiation is focused on the tumor by a machine outsidethe body. Another option is intraoperative electron beam radiationadministered during an operation. Chemotherapy can also be used to treatpancreatic cancer patients. Suitable anti-cancer drugs include, but arenot limited to, 5-fluorouracil (5-FU), mitomycin, ifosfamide,doxorubicin, streptozocin, chlorozotocin, and combinations thereof. Themethods provided by the invention can provide a beneficial effect forpancreatic cancer patients, by administration of a polypeptide of theinvention or a combination of administration of a peptidomimeticmacrocycle and surgery, radiation therapy, or chemotherapy.

In one aspect, peptidomimetic macrocycles of the invention can be usedfor the treatment of colon cancer, including but not limited tonon-neoplastic polyps, adenomas, familial syndromes. colorectalcarcinogenesis, colorectal carcinoma, and carcinoid tumors. Possibletreatments available for colon cancer that can be used in conjunctionwith the peptidomimetic macrocycles of the invention include surgery,chemotherapy, radiation therapy or targeted drug therapy.

Radiation therapy can include external beam radiation therapy and/orbrachytherapy. Chemotherapy can be used to reduce the likelihood ofmetastasis developing, shrink tumor size, or slow tumor growth.Chemotherapy is often applied after surgery (adjuvant), before surgery(neo-adjuvant), or as the primary therapy if surgery is not indicated(palliative). For example, exemplary regimens for adjuvant chemotherapyinvolve the combination of infusional 5-fluorouracil, leucovorin, andoxaliplatin (FOLFOX). First line chemotherapy regimens can involve thecombination of infusional 5-fluorouracil, leucovorin, and oxaliplatin(FOLFOX) with a targeted drug such as bevacizumab, cetuximab orpanitumumab or infusional 5-fluorouracil, leucovorin, and irinotecan(FOLFIRI) with targeted drug such as bevacizumab, cetuximab orpanitumumab. Other chemotherapeutic agents that can be useful in thetreatment or prevention of colon cancer in combination with thepeptidomimetic macrocycles of the invention are Bortezomib (Velcade®),Oblimersen (Genasense®, G3139), Gefitinib and Erlotinib (Tarceva®) andTopotecan (Hycamtin®).

Some embodiments provide methods for the treatment of lung cancer usingthe peptidomimetic macrocycles of the invention. Examples of cellularproliferative and/or differentiative disorders of the lung include, butare not limited to, bronchogenic carcinoma, including paraneoplasticsyndromes, bronchioloalveolar carcinoma, neuroendocrine tumors, such asbronchial carcinoid, miscellaneous tumors, and metastatic tumors:pathologies of the pleura, including inflammatory pleural effusions,noninflammatory pleural effusions. pneumothorax, and pleural tumors,including solitary fibrous tumors (pleural fibroma) and malignantmesothelioma.

The most common type of lung cancer is non-small cell lung cancer(NSCLC), which accounts for approximately 80-85% of lung cancers and isdivided into squamous cell carcinomas, adenocarcinomas, and large cellundifferentiated carcinomas. Small cell lung cancer, e.g. small celllung carcinomas, accounts for 15-20% of lung cancers. Treatment optionsfor lung cancer include surgery, immunotherapy, radiation therapy,chemotherapy, photodynamic therapy, or a combination thereof. Somepossible surgical options for treatment of lung cancer are a segmentalor wedge resection. a lobectomy, or a pneumonectomy. Radiation therapycan be external beam radiation therapy or brachytherapy. Someanti-cancer drugs that can be used in chemotherapy to treat lung cancerin combination with the peptidomimetic macrocycles of the inventioninclude cisplatin, carboplatin, paclitaxel, docetaxel, gemcitabine,vinorelbine, irinotecan, etoposide, vinblastine, gefitinib, ifosfamide,methotrexate, or a combination thereof. Photodynamic therapy (PDT) canbe used to treat lung cancer patients. The methods described herein canprovide a beneficial effect for lung cancer patients, by administrationof a peptidomimetic macrocycle or a combination of administration of apeptidomimetic macrocycle and surgery, radiation therapy, chemotherapy,photodynamic therapy, or a combination thereof.

Examples of cellular proliferative and/or differentiative disorders ofthe liver include, but are not limited to, nodular hyperplasias,adenomas, and malignant tumors, including primary carcinoma of the liverand metastatic tumors.

Immunoproliferative disorders (also known as “immunoproliferativediseases” or “immunoproliferative neoplasms”) are disorders of theimmune system that are characterized by the abnormal proliferation ofthe primary cells of the immune system, which includes B cells, T cellsand Natural Killer (NK) cells, or by the excessive production ofimmunoglobulins (also known as antibodies). Such disorders include thegeneral categories of lymphoproliferative disorders,hypergammaglobulinemias, and paraproteinemias. Examples of suchdisorders include, but are not limited to, X-linked lymphoproliferativedisorder, autosomal lymphoproliferative disorder, Hyper-IgM syndrome,heavy chain disease, and cryoglobulinemia. Other immunoproliferativedisorders can be graft versus host disease (GVHD); psoriasis; immunedisorders associated with graft transplantation rejection; T celllymphoma; T cell acute lymphoblastic leukemia; testicular angiocentric Tcell lymphoma; benign lymphocytic angiitis; and autoimmune diseases suchas lupus erythematosus, Hashimoto's thyroiditis, primary myxedema,Graves' disease, pernicious anemia, autoimmune atrophic gastritis,Addison's disease, insulin dependent diabetes mellitis, good pasture'ssyndrome. myasthenia gravis, pemphigus, Crohn's disease, sympatheticophthalmia, autoimmune uveitis, multiple sclerosis, autoimmune hemolyticanemia, idiopathic thrombocytopenia, primary biliary cirrhosis, chronicaction hepatitis, ulceratis colitis, Sjogren's syndrome, rheumatoidarthritis, polymyositis, scleroderma, and mixed connective tissuedisease.

Combination Treatments

In one embodiment. peptidomimetic macrocycles of the invention can beused for the treatment of cancer in conjunction with alkylating andalkylating-like agents. Such agents include, for example, nitrogenmustards such as chlorambucil, chlormethine, cyclophosphamide,ifosfamide, and melphalan; nitrosoureas such as carmustine, fotemustine,lomustine, and streptozocin; platinum therapeutic agents such ascarboplatin, cisplatin, oxaliplatin, BBR3464, and satraplatin; or otheragents, including but not limited to busulfan, dacarbazine,procarbazine, temozolomide, thiotepa, treosulfan, or uramustine.

In another embodiment, peptidomimetic macrocycles of the invention canbe used in conjunction with an antineoplastic agent which is anantimetabolite. For example, such an antineoplastic agent can be a folicacid such as aminopterin, methotrexate, pemetrexed, or raltitrexed.Alternatively, the antineoplastic agent can be a purine, including butnot limited to cladribine, clofarabine, fludarabine, mercaptopurine,pentostatin, thioguanine. In further embodiments, the antineoplasticagent can be a pyrimidine such as capecitabine, cytarabine,fluorouracil, floxuridine, and gemcitabine.

In still other embodiments, peptidomimetic macrocycles of the inventioncan be used in conjunction with an antineoplastic agent which is anspindle poison/mitotic inhibitor. Agents in this category includetaxanes, for example docetaxel and paclitaxel; and vinca alkaloids suchas vinblastine, vincristine, vindesine, and vinorelbine. In yet otherembodiments, peptidomimetic macrocycles of the invention can be used incombination with an antineoplastic agent which is a cytotoxic/antitumorantibiotic from the anthracycline family such as daunorubicin,doxorubicin, epirubicin, idarubicin, mitoxantrone. pixantrone, orvalrubicin; an antibiotic from the streptomyces family such asactinomycin, bleomycin, mitomycin, or plicamycin; or hydroxyurea.Alternatively, agents used for combination therapy can be topoisomeraseinhibitors including, but not limited to camptothecin, topotecan,irinotecan, etoposide, or teniposide.

Alternatively, the antineoplastic agent can be an antibody orantibody-derived agent. For example, a receptor tyrosine kinase-targetedantibody such as cetuximab, panitumumab, or trastuzumab can be used.Alternatively, the antibody can be an anti-CD20 antibody such asrituximab or tositumomab, or any other suitable antibody including butnot limited to alemtuzumab, bevacizumab, and gemtuzumab. In otherembodiments, the antineoplastic agent is a photosensitizer such asaminolevulinic acid, methyl aminolevulinate, porfimer sodium, orverteporfin. In still other embodiments, the antineoplastic agent is atyrosine kinase inhibitor such as dediranib, dasatinib, erlotinib,gefitinib, imatinib, lapatinib, nilotinib, sorafenib, sunitinib, orvandetanib. Other neoplastic agents suitable in the use of the inventioninclude, for example, alitretinoin, tretinoin, altretamine, amsacrine,anagrelide, arsenic trioxide, asparaginase (pegaspargase), bexarotene,bortezomib, denileukin diftitox, estramustine, ixabepilone, masoprocol,or mitotane.

In other or further embodiments, the peptidomimetics macrocyclesdescribed herein are used to treat, prevent or diagnose conditionscharacterized by overactive cell death or cellular death due tophysiologic insult, etc. Some examples of conditions characterized bypremature or unwanted cell death are or alternatively unwanted orexcessive cellular proliferation include, but are not limited tohypocellular/hypoplastic, acellular/aplastic, orhypercellular/hyperplastic conditions. Some examples include hematologicdisorders including but not limited to fanconi anemia, aplastic anemia,thalaessemia, congenital neutropenia, myclodysplasia

In other or further embodiments, the peptidomimetics macrocycles of theinvention that act to decrease apoptosis are used to treat disordersassociated with an undesirable level of cell death. Thus, in someembodiments, the anti-apoptotic peptidomimetics macrocycles of theinvention are used to treat disorders such as those that lead to celldeath associated with viral infection, e.g., infection associated withinfection with human immunodeficiency virus (HIV). A wide variety ofneurological diseases are characterized by the gradual loss of specificsets of neurons, and the anti-apoptotic peptidomimetics macrocycles ofthe invention are used, in some embodiments, in the treatment of thesedisorders. Such disorders include Alzheimer's disease, Parkinson'sdisease, amyotrophic lateral sclerosis (ALS) retinitis pigmentosa,spinal muscular atrophy, and various forms of cerebellar degeneration.The cell loss in these diseases does not induce an inflammatoryresponse, and apoptosis appears to be the mechanism of cell death. Inaddition, a number of hematologic diseases are associated with adecreased production of blood cells. These disorders include anemiaassociated with chronic disease, aplastic anemia, chronic neutropenia,and the myclodysplastic syndromes. Disorders of blood cell production,such as myelodysplastic syndrome and some forms of aplastic anemia, areassociated with increased apoptotic cell death within the bone marrow.These disorders could result from the activation of genes that promoteapoptosis, acquired deficiencies in stromal cells or hematopoieticsurvival factors, or the direct effects of toxins and mediators ofimmune responses. Two common disorders associated with cell death aremyocardial infarctions and stroke. In both disorders, cells within thecentral area of ischemia, which is produced in the event of acute lossof blood flow, appear to die rapidly as a result of necrosis. However,outside the central ischemic zone, cells die over a more protracted timeperiod and morphologically appear to die by apoptosis.

In other or further embodiments, the anti-apoptotic peptidomimeticsmacrocycles of the invention are used to treat all such disordersassociated with undesirable cell death.

Some examples of immunologic disorders that are treated with thepeptidomimetics macrocycles described herein include but are not limitedto organ transplant rejection, arthritis, lupus, IBD, Crohn's disease,asthma, multiple sclerosis, diabetes, etc.

Some examples of neurologic disorders that are treated with thepeptidomimetics macrocycles described herein include but are not limitedto Alzheimer's Disease, Down's Syndrome, Dutch Type Hereditary CerebralHemorrhage Amyloidosis, Reactive Amyloidosis, Familial AmyloidNephropathy with Urticaria and Deafness, Muckle-Wells Syndrome,Idiopathic Myeloma; Macroglobulinemia-Associated Myeloma, FamilialAmyloid Polyneuropathy, Familial Amyloid Cardiomyopathy, IsolatedCardiac Amyloid, Systemic Senile Amyloidosis, Adult Onset Diabetes,Insulinoma, Isolated Atrial Amyloid, Medullary Carcinoma of the Thyroid,Familial Amyloidosis, Hereditary Cerebral Hemorrhage With Amyloidosis,Familial Amyloidotic Polyneuropathy, Scrapie, Creutzfeldt-Jacob Disease,Gerstmann Straussler-Scheinker Syndrome, Bovine Spongiform Encephalitis,a prion-mediated disease, and Huntington's Disease.

Some examples of endocrinologic disorders that are treated with thepeptidomimetics macrocycles described herein include but are not limitedto diabetes, hypothyroidism, hypopituitarism, hypoparathyroidism,hypogonadism, etc.

Examples of cardiovascular disorders (e.g., inflammatory disorders) thatare treated or prevented with the peptidomimetics macrocycles of theinvention include, but are not limited to, atherosclerosis, myocardialinfarction, stroke, thrombosis, aneurism, heart failure, ischemic heartdisease, angina pectoris, sudden cardiac death, hypertensive heartdisease; non-coronary vessel disease, such as arteriolosclerosis, smallvessel disease, nephropathy, hypertriglyceridemia, hypercholesterolemia,hyperlipidemia, xanthomatosis, asthma, hypertension, emphysema andchronic pulmonary disease; or a cardiovascular condition associated withinterventional procedures (“procedural vascular trauma”), such asrestenosis following angioplasty, placement of a shunt, stent, syntheticor natural excision grafts, indwelling catheter, valve or otherimplantable devices. Preferred cardiovascular disorders includeatherosclerosis, myocardial infarction, aneurism, and stroke.

In some embodiments, the peptidomimetic macrocycles are useful in thetreatment of viral disorders. For example, in the PB1/PA system, labeledpeptidomimetic macrocycles based on PB1 can be used in a PA bindingassay along with small molecules that competitively bind to PA.Competitive binding studies allow for rapid in vitro evaluation anddetermination of drug candidates specific for the PB1/PA system. Suchbinding studies can be performed with any of the peptidomimeticmacrocycles disclosed herein and their binding partners. Antibodies canalso be developed which disrupt the binding between PA and PD1.

For example, peptidomimetic macrocycles derived from the PB1 helixsequence, or peptidomimetic macrocycles that bind selectively to the PB1peptide binding site of the PA protein, can selectively inhibitinfluenza RNA-dependent RNA polymerases. Peptidomimetic macrocyclesderived from the PB2 helix sequence, or peptidomimetic macrocycles thatbind selectively to the PB2 peptide binding site of the PB1 protein, canselectively inhibit influenza RNA-dependent RNA polymerases. Whenadministered within a therapeutic window after infection, suchpeptidomimetic macrocycles can reduce the severity or duration of aninfluenza infection. When administered prophylactically, suchpeptidomimetic macrocycles can prevent infection by influenza virusesand thereby decrease the spread of influenza and reduce large-scaleepidemics.

In one aspect, the present invention provides novel peptidomimeticmacrocycles that are useful in competitive binding assays to identifyagents which bind to the natural ligand(s) of the proteins or peptidesupon which the peptidomimetic macrocycles are modeled. For example, inthe PB1/PA system, labeled peptidomimetic macrocycles based on PB1 canbe used in a PA binding assay along with small molecules thatcompetitively bind to PA. Competitive binding studies allow for rapid invitro evaluation and determination of drug candidates specific for thePB1/PA system. Such binding studies can be performed with any of thepeptidomimetic macrocycles disclosed herein and their binding partners.

In other aspects, the present invention provides for both prophylacticand therapeutic methods of treating a subject infected with, at risk of,or susceptible to an influenza virus. These methods compriseadministering an effective amount of a compound to a warm bloodedanimal, including a human. In some embodiments, the administration ofthe compounds of the present invention prevents the proliferation ortransmission of an influenza virus.

In some embodiments, peptidomimetic macrocycles are used to treatdiseases induced by influenza viruses. Like other viruses, thereplication of influenza virus involves six phases: transmission, entry,replication, biosynthesis, assembly, and exit. Entry occurs byendocytosis, replication and vRNP assembly takes place in the nucleus,and the virus buds from the plasma membrane. In the infected patient,the virus targets airway epithelial cells.

The methods described herein are also useful for development and/oridentification of agents for the treatment of infections caused byviruses such as Abelson leukemia virus. Abelson murine leukemia virus,Abelson's virus, Acute laryngotracheobronchitis virus, Adelaide Rivervirus, Adeno associated virus group, Adenovirus. African horse sicknessvirus, African swine fever virus, AIDS virus, Aleutian mink diseaseparvovirus, Alpharetrovirus, Alphavirus, ALV related virus, Amaparivirus, Aphthovirus, Aquareovirus, Arbovirus, Arbovirus C, arbovirusgroup A, arbovirus group B, Arenavirus group, Argentine hemorrhagicfever virus, Argentine hemorrhagic fever virus, Arterivirus, Astrovirus,Ateline herpesvirus group, Aujezky's disease virus, Aura virus, Ausdukdisease virus. Australian bat lyssavirus, Aviadenovirus, avianerythroblastosis virus, avian infectious bronchitis virus, avianleukemia virus, avian leukosis virus, avian lymphomatosis virus, avianmycloblastosis virus, avian paramyxovirus, avian pneumoencephalitisvirus, avian reticuloendotheliosis virus, avian sarcoma virus, aviantype C retrovirus group, Avihepadnavirus, Avipoxvirus, B virus, B19virus, Babanki virus, baboon herpesvirus, baculovirus, Barmah Forestvirus, Bebaru virus, Berrimah virus, Betaretrovirus, Bimavirus, Bittnervirus, BK virus, Black Creek Canal virus, bluetongue virus, Bolivianhemorrhagic fever virus, Boma disease virus, border disease of sheepvirus, boma virus, bovine alphaherpesvirus 1, bovine alphaherpesvirus 2,bovine coronavirus, bovine ephemeral fever virus, bovineimmunodeficiency virus, bovine leukemia virus, bovine leukosis virus,bovine mammillitis virus, bovine papillomavirus, bovine papularstomatitis virus, bovine parvovirus, bovine syncytial virus, bovine typeC oncovirus. bovine viral diarrhea virus, Buggy Creek virus, bulletshaped virus group. Bunyamwera virus supergroup, Bunyavirus, Burkitt'slymphoma virus, Bwamba Fever, CA virus, Calicivirus, Californiaencephalitis virus, camelpox virus, canarypox virus, canid herpesvirus,canine coronavirus, canine distemper virus, canine herpesvirus, canineminute virus, canine parvovirus, Cano Delgadito virus, caprine arthritisvirus, caprine encephalitis virus, Caprine Herpes Virus, Capripox virus,Cardiovirus, caviid herpesvirus 1, Cereopithecid herpesvirus 1,cercopithecine herpesvirus 1, Cercopithecine herpesvirus 2. Chandipuravirus, Changuinola virus, channel catfish virus, Charleville virus,chickenpox virus, Chikungunya virus, chimpanzee herpesvirus, chubreovirus, chum salmon virus, Cocal virus, Coho salmon reovirus, coitalexanthema virus. Colorado tick fever virus, Coltivirus, Columbia SKvirus, common cold virus, contagious ecthyma virus, contagious pustulardermatitis virus, Coronavirus, Corriparta virus, coryza virus, cowpoxvirus, coxsackie virus, CPV (cytoplasmic polyhedrosis virus), cricketparalysis virus, Crimean-Congo hemorrhagic fever virus, croup associatedvirus, Cryptovirus, Cypovirus, Cytomegalovirus, cytomegalovirus group,cytoplasmic polyhedrosis virus, deer papillomavirus, deltaretrovirus,dengue virus, Densovirus, Dependovirus, Dhori virus, diploma virus,Drosophila C virus, duck hepatitis B virus, duck hepatitis virus 1, duckhepatitis virus 2, duovirus, Duvenhage virus, Deformed wing virus DWV,eastern equine encephalitis virus, eastern equine encephalomyelitisvirus, EB virus, Ebola virus, Ebola-like virus, echo virus, echovirus,echovirus 10, echovirus 28, echovirus 9, ectromelia virus, EEE virus,EIA virus, EIA virus, encephalitis virus, encephalomyocarditis groupvirus, encephalomyocarditis virus, Enterovirus, enzyme elevating virus,enzyme elevating virus (LDH), epidemic hemorrhagic fever virus,epizootic hemorrhagic disease virus, Epstein-Barr virus, equidalphaherpesvirus 1, equid alphaberpesvirus 4, equid herpesvirus 2,equine abortion virus, equine arteritis virus, equine encephalosisvirus, equine infectious anemia virus, equine morbillivirus, equinerhinopneumonitis virus, equine rhinovirus, Eubenangu virus, European elkpapillomavirus, European swine fever virus, Everglades virus, Eyachvirus, felid herpesvirus 1, feline calicivirus, feline fibrosarcomavirus, feline herpesvirus, feline immunodeficiency virus, felineinfectious peritonitis virus, feline leukemia/sarcoma virus, felineleukemia virus, feline panleukopenia virus, feline parvovirus, felinesarcoma virus, feline syncytial virus, Filovirus, Flanders virus,Flavivirus, foot and mouth disease virus, Fort Morgan virus. FourCorners hantavirus, fowl adenovirus 1, fowlpox virus, Friend virus,Gammaretrovirus, GB hepatitis virus, GB virus. German measles virus,Getah virus, gibbon ape leukemia virus, glandular fever virus, goatpoxvirus, golden shinner virus, Gonometa virus, goose parvovirus,granulosis virus. Gross' virus, ground squirrel hepatitis B virus, groupA arbovirus, Guanarito virus, guinea pig cytomegalovirus, guinea pigtype C virus, Hantaan virus, Hantavirus, hard clam reovirus, harefibroma virus, HCMV (human cytomegalovirus), hemadsorption virus 2,hemagglutinating virus of Japan, hemorrhagic fever virus, hendra virus,Henipaviruses, Hepadnavirus, hepatitis A virus, hepatitis B virus group,hepatitis C virus, hepatitis D virus, hepatitis delta virus, hepatitis Evirus, hepatitis F virus, hepatitis G virus, hepatitis nonA nonB virus,hepatitis virus, hepatitis virus (nonhuman), hepatoencephalomyelitisreovirus 3, Hepatovirus, heron hepatitis B virus, herpes B virus, herpessimplex virus, herpes simplex virus 1, herpes simplex virus 2,herpesvirus, herpesvirus 7. Herpesvirus ateles, Herpesvirus hominis,Herpesvirus infection, Herpesvirus saimiri, Herpesvirus suis,Herpesvirus varicellae, Highlands J virus, Hirame rhabdovirus, hogcholera virus, human adenovirus 2, human alphaherpesvirus 1, humanalphaherpesvirus 2, human alphaherpesvirus 3, human B lymphotropicvirus, human betaherpesvirus 5, human coronavirus, human cytomegalovirusgroup, human foamy virus, human gammaherpesvirus 4. humangammaherpesvirus 6, human hepatitis A virus, human herpesvirus 1 group,human herpesvirus 2 group, human herpesvirus 3 group, human herpesvirus4 group, human herpesvirus 6, human herpesvirus 8, humanimmunodeficiency virus, human immunodeficiency virus 1, humanimmunodeficiency virus 2, human papillomavirus, human T cell leukemiavirus, human T cell leukemia virus I, human T cell leukemia virus II,human T cell leukemia virus III. human T cell lymphoma virus I, human Tcell lymphoma virus II, human T cell lymphotropic virus type 1, human Tcell lymphotropic virus type 2, human T lymphotropic virus I, human Tlymphotropic virus II, human T lymphotropic virus III, Ichnovirus,infantile gastroenteritis virus, infectious bovine rhinotracheitisvirus, infectious haematopoietic necrosis virus, infectious pancreaticnecrosis virus, influenza virus A, influenza virus B. influenza virus C.influenza virus D, influenza virus pr8, insect iridescent virus, insectvirus, iridovirus, Japanese B virus, Japanese encephalitis virus, JCvirus, Junin virus, Kaposi's sarcoma-associated herpesvirus, Kemerovovirus, Kilham's rat virus, Klamath virus, Kolongo virus, Koreanhemorrhagic fever virus, kumba virus, Kysanur forest disease virus,Kyzylagach virus, La Crosse virus, lactic dehydrogenase elevating virus,lactic dehydrogenase virus, Lagos bat virus, Langur virus, lapineparvovirus, Lassa fever virus, Lassa virus, latent rat virus, LCM virus,Leaky virus, Lentivirus, Leporipoxvirus, leukemia virus, leukovirus,lumpy skin disease virus, lymphadenopathy associated virus,Lymphocryptovirus, lymphocytic choriomeningitis virus,lymphoproliferative virus group, Machupo virus, mad itch virus,mammalian type B oncovirus group, mammalian type B retroviruses,mammalian type C retrovirus group, mammalian type D retroviruses,mammary tumor virus, Mapuera virus, Marburg virus, Marburg-like virus,Mason Pfizer monkey virus, Mastadenovirus, Canaro virus, ME virus,measles virus. Menangle virus, Mengo virus, Mengovirus, Middelburgvirus, milkers nodule virus, mink enteritis virus, minute virus of mice,MLV related virus, MM virus, Mokola virus, Molluscipoxvirus, Molluscumcontagiosum virus, monkey B virus, monkeypox virus, Mononegavirales,Morbillivirus, Mount Elgon bat virus, mouse cytomegalovirus, mouseencephalomyelitis virus, mouse hepatitis virus, mouse K virus, mouseleukemia virus, mouse mammary tumor virus, mouse minute virus, mousepneumonia virus, mouse poliomyelitis virus, mouse polyomavirus, mousesarcoma virus, mousepox virus, Mozambique virus, Mucambo virus, mucosaldisease virus, mumps virus, murid betaherpesvirus 1, muridcytomegalovirus 2, murine cytomegalovirus group, murineencephalomyelitis virus, murine hepatitis virus, murine leukemia virus,murine nodule inducing virus, murine polyomavirus, murine sarcoma virus,Muromegalovirus, Murray Valley encephalitis virus, myxoma virus,Myxovirus, Myxovirus multiforme, Myxovirus parotitidis, Nairobi sheepdisease virus, Nairovirus, Nanimavirus, Nariva virus, Ndumo virus,Neethling virus, Nelson Bay virus, neurotropic virus, New WorldArenavirus, newborn pneumonitis virus, Newcastle disease virus, Nipahvirus, noncytopathogenic virus, Norwalk virus, nuclear polyhedrosisvirus (NPV), nipple neck virus, O'nyong'nyong virus, Ockelbo virus,oncogenic virus, oncogenic viruslike particle, oncomavirus, Orbivirus,Orf virus, Oropouche virus, Orthohepadnavirus, Orthomyxovirus,Orthopoxvirus, Orthoreovirus, Orungo, ovine papillomavirus, ovinecatarrhal fever virus, owl monkey herpesvirus, Palyam virus,Papillomavirus. Papillomavirus sylvilagi, Papovavirus, parainfluenzavirus, parainfluenza virus type 1, parainfluenza virus type 2,parainfluenza virus type 3, parainfluenza virus type 4, Paramyxovirus,Parapoxvirus, paravaccinia virus, Parvovirus, Parvovirus B19, parvovirusgroup, Pestivirus, Phlebovirus, phocine distemper virus, Picodnavirus,Picomavirus, pig cytomegalovirus, pigeonpox virus, Piry virus, Pixunavirus, pneumonia virus of mice, Pneumovirus, poliomyelitis virus,poliovirus, Polydnavirus, polyhedral virus, polyoma virus, Polyomavirus,Polyomavirus bovis, Polyomavirus cercopitheci, Polyomavirus hominis 2,Polyomavirus maccacae 1. Polyomavirus muris 1, Polyomavirus muris 2,Polyomavirus papionis 1. Polyomavirus papionis 2, Polyomavirussylvilagi, Pongine herpesvirus 1, porcine epidemic diarrhea virus,porcine hemagglutinating encephalomyelitis virus, porcine parvovirus,porcine transmissible gastroenteritis virus, porcine type C virus, poxvirus, poxvirus, poxvirus variolae, Prospect Hill virus, Provirus,pseudocowpox virus, pseudorabies virus, psittacinepox virus, quailpoxvirus, rabbit fibroma virus, rabbit kidney vaculolating virus, rabbitpapillomavirus, rabies virus, raccoon parvovirus, raccoonpox virus,Ranikhet virus, rat cytomegalovirus, rat parvovirus, rat virus,Rauscher's virus, recombinant vaccinia virus, recombinant virus,rovirus, reovirus 1, reovirus 2, reovirus 3, reptilian type C virus,respiratory infection virus, respiratory syncytial virus, respiratoryvirus, reticuloendotheliosis virus, Rhabdovirus. Rhabdovirus carpia,Rhadinovirus, Rhinovirus, Rhizidiovirus, Rift Valley fever virus,Riley's virus, rinderpest virus, RNA tumor virus, Ross River virus,Rotavirus, rougeole virus, Rous sarcoma virus, rubella virus, rubeolavirus, Rubivirus, Russian autumn encephalitis virus, SA 1 I simianvirus, SA2 virus, Sabia virus, Sagiyama virus, Saimirine herpesvirus 1,salivary gland virus, sandfly fever virus group, Sandjimba virus, SARSvirus, SDAV (sialodacryoadenitis virus), sealpox virus, Semliki ForestVirus. Seoul virus, sheeppox virus, Shope fibroma virus, Shope papillomavirus, simian foamy virus, simian hepatitis A virus, simian humanimmunodeficiency virus, simian immunodeficiency virus, simianparainfluenza virus, simian T cell lymphotrophic virus, simian virus,simian virus 40, Simplexvirus, Sin Nombre virus, Sindbis virus, smallpoxvirus. South American hemorrhagic fever viruses, sparrowpox virus,Spumavirus, squirrel fibroma virus, squirrel monkey retrovirus, SSV 1virus group, STLV (simian T lymphotropic virus) type I, STLV (simian Tlymphotropic virus) type II, STLV (simian T lymphotropic virus) typeIII, stomatitis papulosa virus, submaxillary virus, suidalphaherpesvirus 1, suid herpesvirus 2, Suipoxvirus, swamp fever virus,swinepox virus, Swiss mouse leukemia virus, TAC virus, Tacaribe complexvirus, Tacaribe virus, Tanapox virus, Taterapox virus, Tench reovirus,Theiler's encephalomyelitis virus, Theiler's virus, Thogoto virus,Thottapalayam virus. Tick bome encephalitis virus, Tioman virus,Togavirus, Torovirus, tumor virus, Tupaia virus, turkey rhinotracheitisvirus, turkeypox virus, type C retroviruses, type D oncovirus, type Dretrovirus group, ulcerative disease rhabdovirus, Una virus, Uukuniemivirus group, vaccinia virus, vacuolating virus, varicella zoster virus,Varicellovirus, Varicola virus, variola major virus, variola virus,Vasin Gishu disease virus, VEE virus, Venezuelan equine encephalitisvirus, Venezuelan equine encephalomyelitis virus, Venezuelan hemorrhagicfever virus, vesicular stomatitis virus, Vesiculovirus, Vilyuisk virus,viper retrovirus, viral haemorrhagic septicemia virus, Visna Maedivirus, Visna virus, volepox virus, VSV (vesicular stomatitis virus),Wallal virus, Warrego virus, wart virus, WEE virus, West Nile virus,western equine encephalitis virus, western equine encephalomyelitisvirus, Whataroa virus, Winter Vomiting Virus, woodchuck hepatitis Bvirus, woolly monkey sarcoma virus, wound tumor virus, WRSV virus, Yabamonkey tumor virus, Yaba virus, Yatapoxvirus, yellow fever virus, andthe Yug Bogdanovac virus. In one embodiment an infectome will beproduced for each virus that includes an inventory of the host cellulargenes involved in virus infection during a specific phase of viralinfection, such cellular entry or the replication cycle.

For some viruses a great deal of progress has been made in theelucidation of the steps involved during infection of host cells, andany of these steps can be targeted using peptidomimetic macrocycles. Forexample, experiments initiated in the early 1980s showed that influenzavirus follows a stepwise, endocytic entry program with elements sharedwith other viruses such as alpha- and rhabdoviruses (Marsh and Helenius1989; Whittaker 2006). The steps include: 1) Initial attachment tosialic acid containing glycoconjugates receptors on the cell surface; 2)signaling induced by the virus particle; 3) endocytosis byclathrin-dependent and clathrin-independent cellular mechanism; 4)acid-induced, hemaglutinin (HA)-mediated penetration from lateendosomes; 5) acid-activated, M2 and matrix protein (M1) dependentuncoating of the capsid; and, 6) intra-cytosolic transport and nuclearimport of vRNPs. These steps depend on assistance from the host cell inthe form of sorting receptors, vesicle formation machinery,kinase-mediated regulation, organelle acidification, and, most likely,activities of the cytoskeleton.

Influenza attachment to the cells surface occurs via binding of the HA 1subunit to cell surface glycoproteins and glycolipids that carryoligosaccharide moieties with terminal sialic acid residues (Skehel andWiley 2000). The linkage by which the sialic acid is connected to thenext saccharide contributes to species specificity. Avian strainsincluding H5N 1 prefer an a-(2,3)-link and human strains a-(2,6)-link(Matrosovich 2006). In epithelial cells, binding occurs preferentiallyto microvilli on the apical surface, and endocytosis occurs at base ofthese extensions (Matlin 1982). Whether receptor binding induces signalsthat prepare the cell for the invasion is not yet known, but it islikely because activation of protein kinase C and synthesis ofphopshatidylinositol-3-phosphate (P13P) are required for efficient entry(Sieczkarski et al. 2003: Whittaker 2006).

Endocytic internalization occurs within a few minutes after binding(Matlin 1982; Yoshimura and Ohnishi 1984). In tissue culture cellsinfluenza virus makes use of three different types of cellularprocesses; 1) preexisting clathrin coated pits, 2) virus-inducedclathrin coated pits, and 3) endocytosis in vesicles without visiblecoat (Matlin 1982; Sieczkarski and Whittaker 2002; Rust et al. 2004).Video microscopy using fluorescent viruses showed the virus particlesundergoing actin-mediated rapid motion in the cell periphery followed byminus end-directed, microtubule-mediated transport to the perinucleararea of the cell. Live cell imaging indicated that the virus particlesfirst entered a subpopulation of mobile, peripheral early endosomes thatcarry them deeper into the cytoplasm before penetration takes place(Lakadamyali et al. 2003: Rust et al. 2004). The endocytotic process isregulated by protein and lipid kinases, the proteasome, as well as byRabs and ubiquitin-dependent sorting factors (Khor et al. 2003;Whittaker 2006).

The membrane penetration step is mediated by low pH-mediated activationof the trimeric, metastable HA, and the conversion of this Type I viralfusion protein to a membrane fusion competent conformation (Maeda et al.1981; White et al. 1982). This occurs about 16 min after intemalization,and the pH threshold varies between strains in the 5.0-5.6 range. Thetarget membrane is the limiting membrane of intermediate or lateendosomes. The mechanism of fusion has been extensively studied (Kielianand Rey 2006). Further it was observed that fusion itself does not seemto require any host cell components except a lipid bilayer membrane anda functional acidification system (Maeda et al. 1981; White et al.1982). The penetration step is inhibited by agents such aslysosomotropic weak bases, carboxylic ionophores, and proton pumpinhibitors (Matlin 1982: Whittaker 2006).

To allow nuclear import of the incoming vRNPs, the capsid has to bedisassembled. This step involves acidification of the viral interiorthrough the amantadine-sensitive M2-channels causes dissociation of M1from the vRNPs (Bukrinskaya et al. 1982; Martin and Helenius 1991: Pintoet al. 1992). Transport of the individual vRNPs to the nuclear porecomplexes and transfer into the nucleus depends on cellular nucleartransport receptors (O'Neill et al. 1995; Cros et al. 2005). Replicationof the viral RNAs (synthesis of positive and negative strands), andtranscription occurs in complexes tightly associated with the chromatinin the nucleus. It is evident that, although many of the steps arecatalyzed by the viral polymerase, cellular factors are involvedincluding RNA polymerase activating factors, a chaperone HSP90, hCLE,and a human splicing factor UAP56. Viral gene expression is subject tocomplex cellular control at the transcriptional level, a control systemdependent on cellular kinases (Whittaker 2006).

The final assembly of an influenza particle occurs during a buddingprocess at the plasma membrane. In epithelial cells, budding occurs atthe apical membrane domain only (Rodriguez-Boulan 1983). First, theprogeny vRNPs are transported within the nucleoplasm to the nuclearenvelope, then from the nucleus to the cytoplasm, and finally theyaccumulate in the cell periphery. Exit from the nucleus is dependent onviral protein NEP and M1, and a variety of cellular proteins includingCRM1 (a nuclear export receptor), caspases, and possibly some nuclearprotein chaperones. Phosphorylation plays a role in nuclear export byregulating MI and NEP synthesis, and also through the MAPK/ERK system(Bui et al. 1996; Ludwig 2006). G protein and protein kinase signalingis involved in influenza virus budding from infected host cells (Hui E.and Nayak D, 2002).

The three membrane proteins of the virus are synthesized, folded andassembled into oligomers in the ER (Doms et al. 1993). They pass throughthe Golgi complex; undergo maturation through modification of theircarbohydrate moieties and proteolytic cleavage. After reaching theplasma membrane they associate with M1 and the vRNPs in a buddingprocess that result in the inclusion of all eight vRNPs and exclusion ofmost host cell components except lipids.

Influenza infection is associated with activation of several signalingcascades including the MAPK pathway (ERK, JNK, p38 and BMK-1/ERK5), thecB/NF-rcB signaling module, the Raf/MEK/ERK cascade, and programmed celldeath (Ludwig 2006). These result in a variety of effects that limit theprogress of infection such as transcriptional activation of IFN-P,apoptotic cell death, and a block in virus escape of from late endosomes(Ludwig 2006).

Administration

The aqueous pharmaceutical formulations of the present disclosure candraw upon many suitable parenteral modes of administration route. Theformulations can be, for example, administered intravenously,intraarterially, intrathecally, or subcutaneously. If combinations ofagents are administered as separate formulations, they can beadministered by the same route or by different routes.

In some embodiments, the aqueous pharmaceutical formulation isadministered in a single dose. A single dose of the aqueouspharmaceutical formulation can also be used when it is co-administeredwith another substance (e.g., an analgesic) for treatment of an acutecondition.

In some embodiments, the aqueous pharmaceutical formulation (by itselfor in combination with other drugs) is administered in multiple doses.Dosing can be about once, twice, three times, four times, five times,six times, seven times, eight times, nine times, ten times or more thanten times per day. Dosing can be about once a year, twice a year, everysix months, every 4 months, every 3 months, every 60 days, once a month,once every two weeks, once a week, or once every other day. In anotherembodiment the aqueous pharmaceutical formulation alone or incombination with another therapeutic substance is administered togetherabout once per day to about 10 times per day. In another embodiment theadministration of the aqueous pharmaceutical formulation alone or incombination with another therapeutic substance continues for less thanabout 7 days. In yet another embodiment the administration continues formore than about 6, 10, 14, 28 days, two months, six months, or one year.

Administration of the formulations of the disclosure can continue aslong as necessary. In some embodiments, a aqueous pharmaceuticalformulation of the disclosure is administered for more than 1, 2, 3, 4,5, 6, 7, 14, 28, 35, 42, 49, 56, 63, 70, 77, 84, 91, 98, 105, 112, 119,126, 133, or 140 days. In some embodiments, a aqueous pharmaceuticalformulation of the disclosure is administered for less than 140, 133,126, 119, 112, 105, 98, 91, 84, 77, 70, 63, 56, 49, 42, 35, 28, 14, 7,6, 5, 4, 3, 2, or 1 day. In some embodiments, an aqueous pharmaceuticalformulation of the disclosure is administered for more than 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, or 12 days. In some embodiments, a aqueouspharmaceutical formulation of the disclosure is administered for lessthan 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 months. In someembodiments, an aqueous pharmaceutical formulation of the disclosure isadministered for more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12years. In some embodiments, a aqueous pharmaceutical formulation of thedisclosure is administered for less than 12, 11, 10, 9, 8, 7, 6, 5, 4,3, 2, or 1 years. In some embodiments, a aqueous pharmaceuticalformulation of the disclosure is administered chronically on an ongoingbasis.

Dosing for the aqueous pharmaceutical formulation formulations of thedisclosure can be found by routine experimentation. The daily dose canrange from about Ix 10⁴ g to 5000 mg. Daily dose range can depend on theform of the aqueous pharmaceutical formulation e.g., the peptidomimeticmacrocycle used, and/or route of administration, as described herein.For example, daily dose can be in the range of about 0.1-5000 mg, about0.1-3000 mg, about 0.1-2000 mg, about 0.1-1000 mg, about 0.1-500 mg,about 0.1-100 mg, 1-5000 mg, about 1-3000 mg, about 1-2000 mg, about1-1000 mg, about 1-500 mg, or about 1-100 mg, about 10-5000 mg, about10-3000 mg, about 10-2000 mg, about 10-1000 mg, about 10-500 mg, about10-200 mg, about 10-100 mg, about 20-2000 mg, about 20-1500 mg, about20-1000 mg, about 20-500 mg, about 20-100 mg, about 50-5000 mg, about50-4000 mg, about 50-3000 mg, about 50-2000 mg, about 50-1000 mg, about50-500 mg, about 50-100 mg, about 100-5000 mg, about 100-4000 mg, about100-3000 mg, about 100-2000 mg, about 100-1000 mg, about 100-500 mg.

In some embodiments, the daily dose of the aqueous pharmaceuticalformulation is about 0.01, 0.1, 1, 10, 20, 30, 40, 50, 60, 70, 80, 90,100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 mg. In someembodiments, the daily dose of the aqueous pharmaceutical formulation is0.01 mg. In some embodiments, the daily dose of the aqueouspharmaceutical formulation is 0.1 mg. In some embodiments, the dailydose of the aqueous pharmaceutical formulation is 1 mg. In someembodiments, the daily dose of the aqueous pharmaceutical formulation isup to 10 mg. In some embodiments, the daily dose of the aqueouspharmaceutical formulation is up to 20 mg. In some embodiments, thedaily dose of the aqueous pharmaceutical formulation is 50 mg. In someembodiments, the daily dose of the aqueous pharmaceutical formulation is100 mg.

III. Kits

For use in the therapeutic methods of use described herein, theformulations of the disclosure can be available as a kit. Such kits caninclude a carrier, package, or container that is optionallycompartmentalized to receive one or more doses of the aqueouspharmaceutical formulations for use in a method described herein. Thekits provided herein can contain packaging materials. Packagingmaterials for use in packaging pharmaceutical products include, but arenot limited to those described in e.g., U.S. Pat. No. 5,323,907.Examples of pharmaceutical packaging materials include, but are notlimited to, blister packs, bottles, tubes, bags, containers, bottles,and any packaging material suitable for a selected formulation andintended mode of administration and treatment.

The aqueous pharmaceutical formulations of the disclosure can bepackaged in multidose form or in single dose form. In some cases, theformulations are packaged in multidose forms. In some embodiments theformulations are packaged as single dose units. In some embodiments ofthe disclosure single dose packaging of the formulations can offerseveral advantages over multi dose packaging including dosage control,increased patient compliance, improved product labeling, and reducedcounterfeiting. In various embodiments single dosage packaging of theformulations of the disclosure can be in form of vials, ampoules, tubes,bottles, pouches, packets, syringes or blister packs. In someembodiments the single dose containers can be grouped together andplaced into additional containers. In some embodiments the secondarycontainer is a pouch.

In some examples, the formulations of the disclosure can be packed in abottle or a vial. In some examples, the formulations can be packed inglass serum vial. In some examples, the formulations can be packed inserum vials composed of borosilicate glass. In some examples, theformulations are packed in a I mL, a 2 mL, a 3 mL, a 4 mL, a 5 mL, a 10mL, a 20 mL, a 30 mL, or a 50 mL glass vial. In some examples, theformulations are packed in a 5 mL glass vial. In some examples, theformulations are packed in a 10 mL glass vial. In some examples, theformulations are packed in a 15 mL glass vial. In some examples, theformulations are packed in a 20 mL glass vial. In some embodiments, thevials comprise a 5 mm, a 10 mm, a 15 mm, 20 mm, 30 mm, or 50 mm orifice.In some embodiments, the formulations are packed in a 5 mL borosilicateglass vial with a 20 mm orifice. In some embodiments, the formulationsare packed in a 10 mL borosilicate glass vial with a 20 mm orifice. Thecontainers, bottles and/or vials can be equipped with suitable caps orstoppers. In some embodiments, the vials are equipped with a vinylstopper. In some embodiments the formulations are packed in a 10 mLglass vial, with a 20 mm orifice, equipped with vinyl stoppers. Thestoppers can be coated with FluroTek®. The containers, bottles and/orcan also be equipped with a seal, for example, crimped-on flip-off caps.The seal can be aluminum and/or plastic. The container can be a glassampoule.

In some embodiments, the containers, including the vials and thebottles, can be inspected for visible particulates. glass defects,and/or stopper/cap integrity before packaging the formulations therein.In some embodiments, the containers, including the vials and thebottles, can be inspected for visible particulates, glass defects,and/or stopper/cap integrity after packaging the formulations therein.In some embodiments, the containers, including the vials and thebottles, can be inspected for visible particulates, glass defects,and/or stopper/cap integrity before and/or after packaging theformulations therein. The containers, including the vials and thebottles, can also be additionally inspected for fill height afterpackaging the formulations therein. The inspection can be visualinspections and can be carried out under any convenient condition, forexample in front of a black and white background.

A kit can also include labels listing contents and/or instructions foruse, and package inserts with instructions for use. A set ofinstructions will also typically be included. In one embodiment, a labelis on or associated with the container. In one embodiment, a label is ona container when letters, numbers or other characters forming the labelare attached, molded or etched into the container itself; a label isassociated with a container when it is present within a receptacle orcarrier that also holds the container, e.g., as a package insert. In oneembodiment, a label is used to indicate that the contents are to be usedfor a specific therapeutic application. The label also indicatesdirections for use of the contents, such as in the methods describedherein. The labels can optionally indicate one or more items selectedfrom a group comprising the date of manufacturing of the formulation,the recommended storage conditions, intended mode of administration forthe formulation, the amount of formulation enclosed and/or theconcentration of the peptidomimetic macrocycle. The labels can furtherinclude any applicable warnings and/or possible side effects.

In certain embodiments, the pharmaceutical formulations are presented ina pack or dispenser device which contains one or more unit dosage formscontaining a formulation provided herein. The pack, for example,contains metal or plastic foil, such as a blister pack. In oneembodiment, the pack or dispenser device is accompanied by instructionsfor administration. In one embodiment, the pack or dispenser is alsoaccompanied with a notice associated with the container in formprescribed by a governmental agency regulating the manufacture, use, orsale of pharmaceuticals, which notice is reflective of approval by theagency of the form of the drug for human or veterinary administration.Such notice, for example, is the labeling approved by the U.S. Food andDrug Administration for prescription drugs, or the approved productinsert. In one embodiment, formulations containing a compound providedherein formulated in a compatible pharmaceutical carrier are alsoprepared, placed in an appropriate container, and labeled for treatmentof an indicated condition.

EXAMPLES Example 1: Peptidomimetic Macrocycles

Peptidomimetic macrocycles were synthesized, purified and analyzed aspreviously described and as described below (Schafmeister et al., J. Am.Chem. Soc. 122:5891-5892 (2000); Schafmeister & Verdine, J. Am. Chem.Soc. 122:5891 (2005); Walensky et al., Science 305:1466-1470 (2004); andU.S. Pat. No. 7,192,713). Peptidomimetic macrocycles were designed byreplacing two or more naturally occurring amino acids with thecorresponding synthetic amino acids. Substitutions were made at i andi+4, and i and i+7 positions. Peptide synthesis was performed eithermanually or on an automated peptide synthesizer (Applied Biosystems,model 433A), using solid phase conditions, rink amide AM resin(Novabiochem), and Fmoc main-chain protecting group chemistry. For thecoupling of natural Fmoc-protected amino acids (Novabiochem), 10equivalents of amino acid and a 1:1:2 molar ratio of coupling reagentsHBTU/HOBt (Novabiochem)/DIEA were employed. Non-natural amino acids (4equiv) were coupled with a 1:1:2 molar ratio of HATU (AppliedBiosystems)/HOBt/DIEA. The N-termini of the synthetic peptides wereacetylated, while the C-termini were amidated.

Purification of cross-linked compounds was achieved by high performanceliquid chromatography (HPLC) (Varian ProStar) on a reverse phase C18column (Varian) to yield the pure compounds. Chemical composition of thepure products was confirmed by LC/MS mass spectrometry (Micromass LCTinterfaced with Agilent 1100 HPLC system) and amino acid analysis(Applied Biosystems, model 420A).

The following protocol was used in the synthesis of dialkyne-crosslinkedpeptidomimetic macrocycles, including SP662, SP663 and SP664. Fullyprotected resin-bound peptides were synthesized on a PEG-PS resin(loading 0.45 mmol/g) on a 0.2 mmol scale. Deprotection of the temporaryFmoc group was achieved by 3×10 min treatments of the resin boundpeptide with 20% (v/v) piperidine in DMF. After washing with NMP (3×),dichloromethane (3×) and NMP (3×), coupling of each successive aminoacid was achieved with 1×60 min incubation with the appropriatepreactivated Fmoc-amino acid derivative. All protected amino acids (0.4mmol) were dissolved in NMP and activated with HCTU (0.4 mmol) and DIEA(0.8 mmol) prior to transfer of the coupling solution to the deprotectedresin-bound peptide. After coupling was completed, the resin was washedin preparation for the next deprotection/coupling cycle. Acetylation ofthe amino terminus was carried out in the presence of aceticanhydride/DIEA in NMP. The LC-MS analysis of a cleaved and deprotectedsample obtained from an aliquot of the fully assembled resin-boundpeptide was accomplished in order to verifying the completion of eachcoupling. In a typical example, tetrahydrofuran (4 ml) and triethylamine(2 ml) were added to the peptide resin (0.2 mmol) in a 40 ml glass vialand shaken for 10 minutes. Pd(PPh₃)₂Cl₂ (0.014 g, 0.02 mmol) and copperiodide (0.008 g, 0.04 mmol) were then added and the resulting reactionmixture was mechanically shaken 16 hours while open to atmosphere. Thediyne-cyclized resin-bound peptides were deprotected and cleaved fromthe solid support by treatment with TFA/H₂O/TIS (95/5/5 v/v) for 2.5 hat room temperature. After filtration of the resin the TFA solution wasprecipitated in cold diethyl ether and centrifuged to yield the desiredproduct as a solid. The crude product was purified by preparative HPLC.

The following protocol was used in the synthesis of singlealkyne-crosslinked peptidomimetic macrocycles, including SP665. Fullyprotected resin-bound peptides were synthesized on a Rink amide MBHAresin (loading 0.62 mmol/g) on a 0.1 mmol scale. Deprotection of thetemporary Fmoc group was achieved by 2×20 min treatments of the resinbound peptide with 25% (v/v) piperidine in NMP. After extensive flowwashing with NMP and dichloromethane, coupling of each successive aminoacid was achieved with 1×60 min incubation with the appropriatepreactivated Fmoc-amino acid derivative. All protected amino acids (1mmol) were dissolved in NMP and activated with HCTU (1 mmol) and DIEA (1mmol) prior to transfer of the coupling solution to the deprotectedresin-bound peptide. After coupling was completed, the resin wasextensively flow washed in preparation for the nextdeprotection/coupling cycle. Acetylation of the amino terminus wascarried out in the presence of acetic anhydride/DIEA in NMP/NMM. TheLC-MS analysis of a cleaved and deprotected sample obtained from analiquot of the fully assembled resin-bound peptide was accomplished inorder to verifying the completion of each coupling. In a typicalexample, the peptide resin (0.1 mmol) was washed with DCM. Resin wasloaded into a microwave vial. The vessel was evacuated and purged withnitrogen. Molybdenumhexacarbonyl (0.01 eq, Sigma Aldrich 199959) wasadded. Anhydrous chlorobenzene was added to the reaction vessel. Then2-fluorophenol (Ieq, Sigma Aldrich F12804) was added. The reaction wasthen loaded into the microwave and held at 130° C. for 10 minutes.Reaction can need to be pushed a subsequent time for completion. Thealkyne metathesized resin-bound peptides were deprotected and cleavedfrom the solid support by treatment with TFA/H₂OMIS (94/3/3 v/v) for 3 hat room temperature. After filtration of the resin the TFA solution wasprecipitated in cold diethyl ether and centrifuged to yield the desiredproduct as a solid. The crude product was purified by preparative HPLC.

Table 1 shows a list of peptidomimetic macrocycles that were prepared.Table 1a, Table 1b, Table 1c and Table 1d shows a selection ofpeptidomimetic macrocycles. In some embodiments, peptidomimeticmacrocycles exclude peptidomimetic macrocycles shown in Table 2a. Insome embodiments, peptidomimetic macrocycles do not comprise apeptidomimetic macrocycle structure as shown in Table 2a. In otherembodiments, peptidomimetic macrocycles exclude peptidomimeticmacrocycles shown in Table 2b. In some embodiments. the peptidomimeticmacrocycles disclosed herein do not comprise a peptidomimetic macrocyclestructure as shown in Table 2b.

Example 2: Preparation of a Pharmaceutical Formulation 1-LiterPeptidomimetic Formulation Batch

Aileron peptide 1 is formulated as a pharmaceutical formulation. Aileronpeptide 1 is an alpha helical hydrocarbon cross-linked polypeptidemacrocycle, with an amino acid sequence less than 20 amino acids longthat is derived from the transactivation domain of wild type human P53protein and that contains a phenylalanine , a tryptophan and a leucineamino acid in the same positions relative to each other as in thetransactivation domain of wild type human P53 protein. Aileron peptide Ihas a single cross link spanning amino acids in the i to the i+7position of the amino acid sequence and has more than three amino acidsbetween the i+7 position and the carboxyl terminus. Aileron peptide 1binds to human MDM2 and MDM4 and has an observed mass of 950-975 m/c asmeasured by electrospray ionization-mass spectrometry.

For each liter of formulated Aileron peptide 1 the peptides issequentially dissolve in 900 mL of water for injection 182 mg monosodiumphosphate, monohydrate, 2,968 mg disodium phosphate, anhydrous, and 82.2g of D-trehalose. Add 3.0 mL of a 10% (w/w) aqueous solution ofPolysorbate 20. Slowly add 15,000 mg Aileron peptide I divided bypeptide content divided by peptide purity to the solution understirring. E.g., if the peptide content is 94.3% and the peptide purityis 98.2%, 15,000/94.3*100/98./100 or 16,215 mg of bulk- Aileron peptide1 would have to be added. While the peptide is dissolving the pH of thesolution is kept between 7.5 and 7.7 by the addition of 0.1 N sodiumhydroxide.

After all peptide is dissolved, adjust pH of the solution to 7.5 f 0.1with sodium hydroxide and subsequently q.s. with WFI to 1,000 mL. Stirsolution for 5 minutes and then clarify solution by passing it through a0.22-μm PVDF-membrane filter.

Example 3: Sterile Filtration and Fill

The formulated product is filtered through two serial sterilization 0.22μm PVDF membrane filters into a sterile container that is equipped withthe fill needle. The filling process starts after both filters havepassed the post filtration filter integrity test. If one or both filtersdo not pass the post filtration integrity test, the tandem sterilefiltration process is repeated until both filters pass the test.

All vials are inspected for visible particulates, glass faults, fillhigh and stopper/cap integrity in front of a white and black background.Approximately 180 vial containers are then filled per 1-liter batch to alevel of 5.2 mL to 5.7 mL each, with a fill target of 5.5 mL (the labelclaim is 5.0 mL). Fill volume accuracy is verified throughout the fillprocess. The filling machine loaded with vials and stoppers immediatelystoppers each vial after it is filled. Capping occurs in line withfilling and stopping or can occur separately under ISO Class 5 supplyair. Weight check of the contents of the filled vials is performedthroughout the filling process to assure that the vials receive thespecified fill volume. Any rejected vials are discarded.

Example 4: Stability Analysis

To render the stability study more challenging 2 mL vials with 13 mm 0stoppers and a fill volume of 1.0 mL were selected. The smaller vialsize provided a greater surface to volume ratio which would amplify anycontainer/closure effects on product stability. To assure that allsurfaces of vial were challenged, the vials were stored in an invertedposition. The tested storage conditions on inverted vials are: −20° C.,+5° C., +25° C. and +40° C. RH.

The results of this study are depicted in Table 5. There is noappreciable purity loss at storage temperatures between −20° C. and +25°C. over the 6-month test period and only about a 1.8% purity loss of thesample that is stored at 40° C. over the same period. The observed smallbut continuous increase of RRT values between 0.22 and 0.81 in the 40°C. samples attests to both, the excellent detecting power andstability-indicating capability of the RP-HPLC (TFA) method. Thepeptidomimetic macrocycle concentration stayed within an acceptable±4%range over the 6-month period for all samples independent of the storagetemperature.

TABLE 5 Product stability results of Aileron peptide1 formulated at 15mg/mL in 20 mM sodium phosphate, 240 mM trehalose, 300 ppm Polysorbate20 buffer in a 5-fold scaled-down, inverted container/closureconfiguration to amplify any potential product degrading effects by thecontainer/closure surface. Time Points Intial 1 month 2 Months Storage−20° C. −20° C. 5° C. 25° C. 40° C. −20° C. 5° C. 25° C. 40° C. Assay[%]  100%    97%    96%    99%  103%  104%  101%  102% Purity [%] 95.5%95.9% 95.8% 96.1% 96.0% 95.8% 96.2% 96.1% 95.6% Individual Impurities >0.1% ~RRT 0.22 0.08% ~RRT 0.32 0.09% ~RRT 0.47 0.08% ~RRT 0.81 0.08%~RRT 0.84 0.11% 0.06% 0.13% ~RRT 0.88 0.17% 0.12% 0.12% 0.13% 0.13%0.14% 0.14% 0.14% ~RRT 0.90 0.22% 0.16% 0.19% 0.18% 0.18% 0.16% 0.15%0.20% 0.20% ~RRT 0.93 0.54% 0.47% 0.50% 0.51% 0.44% ~RRT 1.03 2.24%2.21% 2.22% 2.20% 2.20% 2.18% 2.13% 2.27% 2.27% ~RRT 1.04 0.17% 0.11%0.12% 0.17% 0.17% 0.15% 0.13% ND ND ~RRT 1.06 0.10% 0.10% 0.14% 0.14%~RRT 1.07 0.12% 0.13% 0.15% 0.16% 0.16% 0.11% 0.07% 0.14% 0.14% ~RRT1.10 0.95% 0.95% 0.93% 0.93% 0.99% 0.98% 0.89% 1.00% 1.06% ParticulateMatter [Number of particles per container) <10 μm <1 <1 <1 <1 <1 >10 μm140 38 23 28 >25 μm 33 7 6 5 >50 μm 4 2 3 2 Time Points 3 months 6months Storage −20° C. 5° C. 25° C. 40° C. −20° C. 5° C. 25° C. 40° C.Assay [%]    99%    99%    99%    97%  100%    99%    99%    96% Purity[%] 95.9% 95.8% 96.0% 95.2% 95.7% 95.5% 95.7% 93.9% IndividualImpurities > 0.1% ~RRT 0.22 0.08% 0.16% ~RRT 0.32 0.14% 0.33% ~RRT 0.470.14% 0.25% ~RRT 0.81 0.12% 0.25% ~RRT 0.84 0.14% 0.25% ~RRT 0.88 0.13%0.13% 0.14% 0.17% 0.09% 0.18% ~RRT 0.90 0.18% 0.17% 0.21% 0.21% 0.19%0.21% 0.20% 0.23% ~RRT 0.93 0.50% 0.51% 0.55% 0.54% 0.49% 0.52% ~RRT1.03 2.23% 2.24% 2.40% 2.40% 2.15% 2.24% 2.21% 2.30% ~RRT 1.04 0.14%0.14% 0.19% 0.15% 0.12% 0.16% ~RRT 1.06 0.00% 0.00% 0.16% 0.16% 0.09%0.09% 0.13% 0.13% ~RRT 1.07 0.15% 0.15% 0.17% 0.17% 0.12% 0.18% 0.12%0.16% ~RRT 1.10 0.95% 0.96% 0.99% 1.08% 0.92% 0.97% 0.99% 1.20%Particulate Matter [Number of particles per container) <10 μm <2 <1 <1~8 >10 μm 31 >25 μm 3 >50 μm 0 RRT = relative retention time.

Example 5: Comparative Data for TRIS and Phosphate Buffers

Composition of Formulations

Two formulations, F1 and F2 were formulated. Table 6 shows thecompositions of the two formulations. Formulations were filled into 6mL, 0 20 mm, colorless vials. The vials were equipped with teflonserum-stoppers D777-1, ø20 mm and aluminum caps without PP-cap, 0 20 mm,6 vials of each formulation were prepared. The vials were stored at 2-8°C. Exposure to direct sunlight was avoided.

TABLE 6 Composition of formulations F1 and F2: Formu- Peptidomimetriclation Buffer Surfactant Peptide Macrocycle Code system Excipient %(mg/ml) Aileron peptide F1 20 mM 240 mM 0.03 15 1 Na-Phosphate TrehalosepH 7.5 F2 20 mM Tris 0.03 pH 7.5

Two placebo formulations P1 (with 20 mM Na-phosphate buffer) and P2(with 20 mM Tris buffer), without the peptidomimetic macrocycle, werealso prepared.

Every filled 6 ml vial was visually inspected using Seidenader. Theresults of this observation are summarized in Table 7. The observationimages are shown in FIG. 2 .

TABLE 7 Comparative visual inspection of F1, F2, P1, and P2 indicatethat the resulting formulations were comparable and yielded minimalvisible particles. Form. total filled/ no. of vials with particles Codeinspected 0 1-5 6-10 >10 notes F1 6 4 2 1x faster 1x particle F2 6 6 P16 5 1 1x faster P2 6 6

Example 6: Shelf Life of the Peptidomimetic Macrocycles of the Invention

A pharmaceutical formulation of Aileron peptide C, was formulated asdescribed above and stored at varying temperatures (−20° C., 2-8° C.,25° C. 60% humidity and 40° C. 75% humidity. The purity of the sampleswas analyzed at regular time intervals. The results of these experimentsfor Aileron peptide 1 are summarized in FIG. 4 . These experimentssupport a greater than 2 year shelf life at −20° C. and 2-8° C.

Example 7: Stability Testing of Aileron Peptide 1 was Performed on aPharmaceutical Formulation formulated as described above and stored atvarying temperatures (−20° C., 2-8° C., 25° C. 60% humidity and 40° C.75% humidity. The purity of the samples was analyzed at regular timeintervals. The results of these experiments for Aileron peptide I aresummarized in Tables 8, 9, 10, and 11.

TABLE 8 Storage: −20° C. Initial 1 Month 2 Months 3 Months 6 Months 9Months 12 Months Appearance¹ Conforms Conforms Conforms ConformsConforms Conforms Conforms pH² 7.6 7.6 7.6 7.6 7.6 7.6* 7.6 Assay 15.615.6 15.9 15.6 15.4 15.5 15.6 [mg/mL] Purity [%]³ 99.0 99.0 98.9 98.998.9 99.0 99.0 Impurities 0.4% @ 0.4% @ 0.4% @ 0.4% @ 0.4% @ 0.4% @ 0.4%@ [%]⁴ 0.89 0.89 0.89 0.89 0.89 0.88 0.88 0.4% @ 0.4% @ 0.5% @ 0.5% @0.5% @ 0.4% @ 0.4% @ 0.91 0.91 0.91 0.91 0.91 0.91 0.91 0.2% @ 0.2% @0.2% @ 0.2% @ 0.2% @ 0.2% @ 0.2% @ 1.02 1.02 1.02 1.02 1.02 1.02 1.02Particulate 184 18 557 Matter⁵ CCIT⁶ 63 0 8 Endotoxin⁷ conforms conforms<1.8 <1.6 EU/mL EU/mL

TABLE 9 Storage: +5° C. Initial 1 Month 2 Months 3 Months 6 Months 9Months 12 Months Appearance¹ Conforms Conforms Conforms ConformsConforms Conforms Conforms pH² 7.6 7.6 7.6 7.7 7.7 7.6 7.6 Assay 15.615.7 15.8 15.8 15.5 15.6 15.6 [mg/mL] Purity [%]³ 99.0 99.0 99.0 99.098.9 99.0 99.0 Impurities 0.4% @ 0.4% @ 0.4% @ 0.4% @ 0.4% @ 0.4% @ 0.4%@ [%]⁴ 0.89 0.89 0.89 0.89 0.89 0.89 0.89 0.4% @ 0.4% @ 0.4% @ 0.4% @0.5% @ 0.4% @ 0.4% @ 0.91 0.91 0.91 0.91 0.91 0.91 0.91 0.2% @ 0.2% @0.2% @ 0.2% @ 0.2% @ 0.2% @ 0.2% @ 1.02 1.02 1.02 1.02 1.02 1.02 1.02Particulate 184 1 37 171 Matters⁵ CCIT⁶ 63 4 11 Endotoxin⁷ conformsconforms <1.8 <1.6 EU/mL EU/mL

TABLE 10 Storage: +25° C./60% RH Initial 1 Month 2 Months 3 Months 6Months 9 Months 12 Months Appearance¹ Conforms Conforms ConformsConforms Conforms Conforms Conforms pH² 7.6 7.65 7.6 7.6 7.7 7.6 7.6Assay 15.6 15.6 15.8 15.4 15.2 15.1 15.0 [mg/mL] Purity [%]³ 99.0 99.098.9 98.7 98.4 98.4 97.9 Impurities [%]⁴ 0.2% @ 0.3% @ 0.4% @ 0.5% @0.23 0.23 0.23 0.23 0.2% @ 0.32 0.1% @ 0.4 @ 0.82 0.82 0.4% @ 0.4% @0.4% @ 0.4% @ 0.4% @ 0.4% @ 0.4% @ 0.89 0.89 0.89 0.89 0.89 0.89 0.880.4% @ 0.4% @ 0.4% @ 0.4% @ 0.4% @ 0.4% @ 0.4% @ 0.91 0.91 0.91 0.910.91 0.91 0.91 0.2% @ 0.2% @ 0.2% @ 0.2% @ 0.2% @ 0.2% @ 0.2% @ 1.021.02 1.02 1.02 1.02 1.02 1.02 Particulate 184 5 185 Matter⁵ CCIT⁶ 63 1 8Endotoxin⁷ conforms conforms <1.8 <1.6 EU/mL EU/mL ¹Visual appearanceSpecification: Upon thawing, clear, colorless, particulate-free solution²pH Specification: 7.3 to 7.7 ³Purity by RP-HPLC(TFA) Specification:≥95%; no single impurity >3% ⁴>0.1% Impurities listed by RRT ⁵USP <788>(light obstruction) Specification: ≤6,000 particles ≥10 μm and ≤600particles ≥25 μm per vial (upper cell: ≥10 μm particles; lower cells:≥25 μm particles ⁶CCIT Specification: No dye intrusion ⁷Endotoxin: ≤4.4EU/mL (based upon maximum patient dose of 17 mg ALRN-6924 per Kg ofpatient weight)

TABLE 11 Storage: +40° C./75% RH Initial 1 Month 2 Months 3 Months 6Months 9 Months 12 Months Appearance¹ conforms conforms conforms OOS*OOS** OOS** OOS** pH² 7.6 7.64 7.6 7.6 7.6 7.5 7.5 Assay 15.6 15.6 15.415.1 14.6 14.4 14.1 [mg/mL] Purity [%]³ 99.0 98.8 98.6 98.1 97.2 96.295.9 Impurities: 0.3 @ 01.9 0.3 @ 01.9 0.3 @ 01.9 [%]⁴ 0.2 @ 020 0.2 @020 n.d. 0.2% @ 0.2% @ 0.2% @ 0.4% @ 0.3% @ 0.3% @ 0.23 0.23 0.23 0.230.23 0.23 0.3% @ 0.3% @ 0.24 024 0.2% @ 0.2% @ 0.29 0.29 0.2% @ 0.2% @0.2% @ 0.3% @ 0.3% @ 0.34 0.34 0.34 0.33 0.32 0.2% @ 0.36 0.2% @ 0.2% @0.2% @ 0.48 0.47 0.47 0.2% @ 0.2% @ 0.74 0.74 0.2% @ 0.3% @ 0.3% @ 0.4%@ 0.83 0.83 0.82 0.82 0.4% @ 0.4% @ 0.4% @ 0.4% @ 0.4% @ 0.4% @ 0.4% @0.89 0.89 0.89 0.89 0.89 0.88 0.88 0.4% @ 0.4% @ 0.4% @ 0.4% @ 0.4% @0.4% @ 0.4% @ 0.91 0.91 0.91 0.91 0.91 0.91 0.91 0.2% @ 0.2% @ 0.2% @0.3% @ 0.4% @ 0.5% @ 1.02 1.02 1.02 1.02 1.04 1.02 0.2% @ 0.4% @ 0.4% @0.5% @ 1.10 1.10 1.10 1.11 OOS*: Solution turned yellow OOS**: Solutionturned yellow (nonconformance confirmed)

Example 8: Stability Testing of Aileron Peptide 1

Aileron peptide 1 was performed on a pharmaceutical formulationformulated at a concentration of 15 mg/ml, (20 mM sodium phosphate, 240mM D-Trehalose, 330 ppm polysorbate 20. pH 7.5), 5 mL of thisformulation was stored in a 10 ml, clear serum vial (20 mmFluroTectK-coated stopper; 20 mm Flip-Off™ seal). The formulation wasstored at −15° C. and was tested at regular intervals. The results ofthis analysis are summarized in Table 12.

TABLE 12 Attribute Test Specification Results Appearance Upon thawing:Clear, Conforms colorless, particulate- free solution Identity PH 7.5 ±0.2 7.6 Osmolality 220 to 400 mOsmol/Kg 327 RP-HPLC (TFA) Co-elutes withALRN-6924 Conforms reference standard Potency qRP-HPLC (TFA) 15 ± 1.5mg/mL 15.6 Purity RP-HPLC(TFA) Purity ≥95% by 99.0% area integration nosingle impurity >3%¹ RRT 0.88: 0.4% RRT 0.91: 0.4% RRT 1.02: 0.2% SafetyParticulate matter ≥10 μm: ≤6,000 ≥10 μm: USP <788> particulates/vial557 ≥25 μm: ≤600 ≥25 μm: particulates/vial 8 Endotoxin, ≤4.4 EU/mL² <1.6EU/mL USP <85> Container/Closure No dye intrusion Conforms IntegritySterility; No growth No growth USP <71> (Membrane Filtration)¹Impurities >0.1% are listed based on their relative retention time(RRT) with respect to the Aileron peptide 1peptide peak. ²Based on amaximum patient dose of 17 mg Aileron peptide 1 per Kg of patientweight.

Example 9: Stability Testing of Multiple Batches of Aileron Peptide 1Under Varying Storage Conditions

Samples 1-7 of Aileron peptide 1 were formulated at a concentration of15 mg/mL (20 mM sodium phosphate, 240 mM D-Trehalose, 330 ppmpolysorbate 20, pH 7.5). These samples were stored under differentstorage conditions as described in Table 13. The formulations weretested for appearances and purity. The results are summarized in Tables14-16 below.

TABLE 13 Sample Number Sample Description Sample 1 Aileron peptide 1Drug product configuration Upright 15 mg/mL 12 month −20° C. (/−5° C.)Sample 2 Aileron peptide 1 Drug product configuration Upright StorageOnly/Return 15 mg/mL 12 month −20° C. (+/−5° C.) Sample 3 Aileronpeptide 1 Drug product configuration Inverted Storage Only/Return 15mg/mL 12 month 25° C./60% RH (+/−2° C./+/−5% RH) Sample 4 Aileronpeptide 1Drug product configuration Inverted 15 mg/mL 12 month 25°C./60% RH (+/−2° C./+/−5% RH) Sample 5 Aileron peptide 1Drug productconfiguration Inverted Storage Only/Return 15 mg/mL 12 month 5° C.(+/−3° C.) Sample 6 Aileron peptide 1Drug product configuration Inverted15 mg/mL 12 month 5° C. (+/−3° C.) Sample 7 Aileron peptide 1Drugproduct configuration Inverted 15 mg/mL 12 month 40° C./75%RH (+/−2°C./+/−5% RH)

TABLE 14 Results of analysis of sample 1. Test Acceptance Criteria TestResult(s) Appearance Upon thawing: Clear, colorless, Clear, colorless,particulate-free particulate-free solution solution. Meets acceptancecriteria. pH 7.3 to 7.7 7.63 Meets acceptance criteria. RP_HPLC (TFA)for Aileron ≥13.5 mg/mL; ≤16.5 mg/mL Injection #1 = 15.58 mg/mLpeptide-1Concentration Injection #2 = 15.54 mg/mL Mean = 15.6 mg/mLMeets acceptance criteria. RP_HPLC (TFA) Purity by Area ≥95% forALRN-6924 peak Injection #1 = 98.6% Integration Injection #2 = 98.6%Mean = 98.6% Meets acceptance criteria. RP_HPLC (TFA) Purity by Area Nosingle impurity >3% Injection #1 = 0.41% Integration for: LargestImpurity Injection #2 = 0.42% (RRT 0.91) Mean = 0.41% Meets acceptancecriteria. RP_HPLC (TFA) Purity by Area Report Result ≥0.1% Injection #1= 0.07% Integration for: Impurity (RRT Injection #2 = 0.07% 0.87) Mean =0.07% RP_HPLC (TFA) Purity by Area Report Result ≥0.1% Injection #1 =0.39% Integration for: Impurity (RRT Injection #2 = 0.39% 0.88) Mean =0.39% RP_HPLC (TFA) Purity by Area Report Result ≥0.1% Injection #1 =0.11% Integration for: Impurity (RRT Injection #2 = 0.10% 0.94) Mean =0.11% RP_HPLC (TFA) Purity by Area Report Result ≥0.1% Injection #1 =0.16% Integration for: Impurity (RRT Injection #2 = 0.14% 1.02) Mean =0.15% RP_HPLC (TFA) Purity by Area Report Result ≥0.1% Injection #1 =0.09% Integration for: Impurity (RRT Injection #2 = 0.09% 1.04) Mean =0.09% RP_HPLC (TFA) Purity by Area Report Result ≥0.1% Injection #1 =0.07% Integration for: Impurity (RRT Injection #2 = 0.07% 1.10) Mean =0.07% Total Impurities Information 1.3%

TABLE 15 Results of analysis of sample 4. Test Acceptance Criteria TestResult(s) Appearance Upon thawing: Clear, colorless, Clear, colorless,particulate-free particulate-free solution solution. Meets acceptancecriteria. pH 7.3 to 7.7 7.64 Meets acceptance criteria. RP_HPLC (TFA)for: Aileron ≥13.5 mg/mL; ≤16.5 mg/mL Injection #1 = 14.98 mg/mLpeptide-1TEConcentration Injection #2 = 15.00 mg/mL Mean = 15.0 mg/mLMeets acceptance criteria. RP_HPLC (TFA) Purity by Area ≥95% for Aileronpeptide-1 peak Injection #1 = 96.3% Integration Injection #2 = 96.3%Mean = 96.3% Meets acceptance criteria. RP_HPLC (TFA) Purity by Area Nosingle impurity >3% Injection #1 = 0.47% Integration for: LargestImpurity Injection #2 = 0.47% (RRT 0.23) Mean = 0.47% Meets acceptancecriteria. RP_HPLC (TFA) Purity by Area Report Result ≥0.1% Injection #1= 0.11% Integration for: Impurity (RRT Injection #2 = 0.11% 0.16) Mean =0.11% RP_HPLC (TFA) Purity by Area Report Result ≥0.1% Injection #1 =0.13% Integration for: Impurity (RRT Injection #2 = 0.13% 0.19) Mean =0.13% RP_HPLC (TFA) Purity by Area Report Result ≥0.1% Injection #1 =0.07% Integration for: Impurity (RRT Injection #2 = 0.07% 0.22) Mean =0.07% RP_HPLC (TFA) Purity by Area Report Result ≥0.1% Injection #1 =0.10% Integration for: Impurity (RRT Injection #2 = 0.10% 0.24) Mean =0.10% RP_HPLC (TFA) Purity by Area Report Result ≥0.1% Injection #1 =0.07% Integration for: Impurity (RRT Injection #2 = 0.06% 0.25A) Mean =0.06% RP_HPLC (TFA) Purity by Area Report Result ≥0.1% Injection #1 =0.06% Integration for: Impurity (RRT Injection #2 = 0.05% 0.25B) Mean =0.06% RP_HPLC (TFA) Purity by Area Report Result ≥0.1% Injection #1 =0.15% Integration for: Impurity (RRT Injection #2 = 0.11% 0.28) Mean =0.13% RP_HPLC (TFA) Purity by Area Report Result ≥0.1% Injection #1 =0.11% Integration for: Impurity (RRT Injection #2 = 0.09% 0.29) Mean =0.10% RP_HPLC (TFA) Purity by Area Report Result ≥0.1% Injection #1 =0.07% Integration for: Impurity (RRT Injection #2 = 0.06% 0.31) Mean =0.06% RP_HPLC (TFA) Purity by Area Report Result ≥0.1% Injection #1 =0.21% Integration for: Impurity (RRT Injection #2 = 0.20% 0.32) Mean =0.20% RP_HPLC (TFA) Purity by Area Report Result ≥0.1% Injection #1 =0.14% Integration for: Impurity (RRT Injection #2 = 0.13% 0.36) Mean =0.13% RP_HPLC (TFA) Purity by Area Report Result ≥0.1% Injection #1 =0.07% Integration for: Impurity (RRT Injection #2 = 0.08% 0.39) Mean =0.08% RP_HPLC (TFA) Purity by Area Report Result ≥0.1% Injection #1 =0.13% Integration for: Impurity (RRT Injection #2 = 0.14% 0.41) Mean =0.14% RP_HPLC (TFA) Purity by Area Report Result ≥0.1% Injection #1 =0.12% Integration for: Impurity (RRT Injection #2 = 0.14% 0.47) Mean =0.13% RP_HPLC (TFA) Purity by Area Report Result ≥0.1% Injection #1 =0.35% Integration for: Impurity (RRT Injection #2 = 0.37% 0.82) Mean =0.36% RP_HPLC (TFA) Purity by Area Report Result ≥0.1% Injection #1 =0.07% Integration for: Impurity (RRT Injection #2 =0.07% 0.87) Mean =0.07% RP_HPLC (TFA) Purity by Area Report Result ≥0.1% Injection #1 =0.37% Integration for: Impurity (RRT Injection #2 = 0.35% 0.88) Mean =0.36% RP_HPLC (TFA) Purity by Area Report Result ≥0.1% Injection #1 =0.36% Integration for: Impurity (RRT Injection #2 = 0.35% 0.91) Mean =0.36% RP_HPLC (TFA) Purity by Area Report Result ≥0.1% Injection #1 =0.21% Integration for: Impurity (RRT Injection #2 = 0.22% 1.02) Mean =0.22% RP_HPLC (TFA) Purity by Area Report Result ≥0.1% Injection #1 =0.10% Integration for: Impurity (RRT Injection #2 = 0.10% 1.04) Mean =0.10% RP_HPLC (TFA) Purity by Area Report Result ≥0.1% Injection #1 =0.08% Integration for: Impurity (RRT Injection #2 = 0.09% 1.10) Mean =0.09% RP_HPLC (TFA) Purity by Area Report Result ≥0.1% Injection #1 =0.07% Integration for: Impurity (RRT Injection #2 = 0.08% 1.11) Mean =0.07% Total Impurities Information 3.6%

TABLE 16 Results of analysis of sample 6. Test Acceptance Criteria TestResult(s) Appearance Upon thawing: Clear, colorless, Clear, colorless,particulate-free particulate-free solution solution. Meets acceptancecriteria. pH 7.3 to 7.7 7.64 Meets acceptance criteria. RP_HPLC (TFA)for Aileron ≥13.5 mg/mL; ≤16.5 mg/mL Injection #1 = 15.61 mg/mLpeptide-1 Concentration Injection #2 = 15.62 mg/mL Mean = 15.6 mg/mLMeets acceptance criteria. RP_HPLC (TFA) Purity by Area ≥95% for Aileronpeptide-1 peak Injection #1 = 98.6% Integration Injection #2 = 98.6%Mean = 98.6% Meets acceptance criteria. RP_HPLC (TFA) Purity by Area Nosingle impurity >3% Injection #1 = 0.43% Integration for: LargestImpurity Injection #2 = 0.41% (RRT 0.91) Mean = 0.42% Meets acceptancecriteria. RP_HPLC (TFA) Purity by Area Report Result ≥0.1% Injection #1= 0.10% Integration for: Largest Impurity Injection #2 = 0.10% (RRT0.82) Mean = 0.10% RP_HPLC (TFA) Purity by Area Report Result ≥0.1%Injection #1 = 0.08% Integration for: Largest Impurity Injection #2 =0.06% (RRT 0.87) Mean = 0.07% RP_HPLC (TFA) Purity by Area Report Result≥0.1% Injection #1 = 0.39% Integration for: Largest Impurity Injection#2 = 0.40% (RRT 0.88) Mean = 0.40% RP_HPLC (TFA) Purity by Area ReportResult ≥0.1% Injection #1 = 0.08% Integration for: Largest ImpurityInjection #2 = 0.11% (RRT 0.94) Mean = 0.10% RP_HPLC (TFA) Purity byArea Report Result ≥0.1% Injection #1 = 0.15% Integration for: LargestImpurity Injection #2 = 0.14% (RRT 1.02) Mean = 0.14% RP_HPLC (TFA)Purity by Area Report Result ≥0.1% Injection #1 = 0.06% Integration for:Largest Impurity Injection #2 = 0.09% (RRT 1.04) Mean = 0.07% RP_HPLC(TFA) Purity by Area Report Result ≥0.1% Injection #1 = 0.07%Integration for: Largest Impurity Injection #2 = 0.08% (RRT 1.10) Mean =0.08% Total Impurities Information 1.4%

TABLE 17 Results of analysis of sample 7. Test Acceptance Criteria TestResult(s) Appearance Upon thawing: Clear, colorless, Clear, yellow,particulate-free particulate-free solution solution. Does not meetacceptance criteria. pH 7.3 to 7.7 7.45 Meets acceptance criteria.RP_HPLC (TFA) for Aileron ≥13.5 mg/mL; ≤16.5 mg/mL Injection #1 = 14.09mg/mL peptide-1 Concentration Injection #2 = 14.05 mg/mL Mean = 14.1mg/mL Meets acceptance criteria. RP_HPLC (TFA) Purity by Area ≥95% forAileron peptide-1 peak Injection #1 = 94.3% Integration Injection #2 =94.3% Mean = 94.3% Does not meet acceptance criteria. RP_HPLC (TFA)Purity by Area No single impurity >3% Injection #1 = 0.58% Integrationfor: Largest Impurity Injection #2 = 0.59% (RRT 1.02) Mean = 0.59% Meetsacceptance criteria. RP_HPLC (TFA) Purity by Area Report Result ≥0.1%Injection #1 = 0.07% Integration for: Largest Impurity Injection #2 =0.07% (RRT 0.11) Mean = 0.07% RP_HPLC (TFA) Purity by Area Report Result≥0.1% Injection #1 = 0.07% Integration for: Largest Impurity Injection#2 = 0.07% (RRT 0.12) Mean = 0.07% RP_HPLC (TFA) Purity by Area ReportResult ≥0.1% Injection #1 = 0.08% Integration for: Largest ImpurityInjection #2 = 0.08% (RRT 0.16) Mean = 0.08% RP_HPLC (TFA) Purity byArea Report Result ≥0.1% Injection #1 = 0.39% Integration for: LargestImpurity Injection #2 = 0.39% (RRT 0.19A) Mean = 0.39% RP_HPLC (TFA)Purity by Area Report Result ≥0.1% Injection #1 = 0.12% Integration for:Largest Impurity Injection #2 = 0.12% (RRT 0.19B) Mean = 0.12% RP_HPLC(TFA) Purity by Area Report Result ≥0.1% Injection #1 = 0.15%Integration for: Largest Impurity Injection #2 = 0.15% (RRT 0.20) Mean =0.15% RP_HPLC (TFA) Purity by Area Report Result ≥0.1% Injection #1 =0.26% Integration for: Largest Impurity Injection #2 = 0.26% (RRT 0.23)Mean = 0.26% RP_HPLC (TFA) Purity by Area Report Result ≥0.1% Injection#1 = 0.27% Integration for: Largest Impurity Injection #2 = 0.27% (RRT0.24) Mean = 0.27% RP_HPLC (TFA) Purity by Area Report Result ≥0.1%Injection #1 = 0.15% Integration for: Largest Impurity Injection #2 =0.14% (RRT 0.25) Mean = 0.14% RP_HPLC (TFA) Purity by Area Report Result≥0.1% Injection #1 = 0.10% Integration for: Largest Impurity Injection#2 = 0.09% (RRT 0.28) Mean = 0.09% RP_HPLC (TFA) Purity by Area ReportResult ≥0.1% Injection #1 = 0.17% Integration for: Largest ImpurityInjection #2 = 0.18% (RRT 0.29) Mean = 0.17% RP_HPLC (TFA) Purity byArea Report Result ≥0.1% Injection #1 = 0.30% Integration for: LargestImpurity Injection #2 = 0.30% (RRT 0.32) Mean = 0.30% RP_HPLC (TFA)Purity by Area Report Result ≥0.1% Injection #1 = 0.16% Integration for:Largest Impurity Injection #2 = 0.15% (RRT 0.36) Mean = 0.16% RP_HPLC(TFA) Purity by Area Report Result ≥0.1% Injection #1 = 0.14%Integration for: Largest Impurity Injection #2 = 0.14% (RRT 0.41) Mean =0.14% RP_HPLC (TFA) Purity by Area Report Result ≥0.1% Injection #1 =0.06% Integration for: Largest Impurity Injection #2 = 0.06% (RRT 0.43)Mean = 0.06% RP_HPLC (TFA) Purity by Area Report Result ≥0.1% Injection#1 = 0.19% Integration for: Largest Impurity Injection #2 = 0.18% (RRT0.47) Mean = 0.18% RP_HPLC (TFA) Purity by Area Report Result ≥0.1%Injection #1 = 0.23% Integration for: Largest Impurity Injection #2 =0.23% (RRT 0.74) Mean = 0.23% RP_HPLC (TFA) Purity by Area Report Result≥0.1% Injection #1 = 0.35% Integration for: Largest Impurity Injection#2 = 0.36% (RRT 0.82) Mean = 0.35% RP_HPLC (TFA) Purity by Area ReportResult ≥0.1% Injection #1 = 0.09% Integration for: Largest ImpurityInjection #2 = 0.09% (RRT 0.83) Mean = 0.09% RP_HPLC (TFA) Purity byArea Report Result ≥0.1% Injection #1 = 0.07% Integration for: LargestImpurity Injection #2 = 0.06% (RRT 0.87) Mean = 0.07% RP_HPLC (TFA)Purity by Area Report Result ≥0.1% Injection #1 = 0.41% Integration for:Largest Impurity Injection #2 = 0.41% (RRT 0.88) Mean = 0.41% RP_HPLC(TFA) Purity by Area Report Result ≥0.1% Injection #1 = 0.43%Integration for: Largest Impurity Injection #2 = 0.43% (RRT 0.91) Mean =0.43% RP_HPLC (TFA) Purity by Area Report Result ≥0.1% Injection #1 =0.06% Integration for: Largest Impurity Injection #2 = 0.06% (RRT 0.96)Mean = 0.06% RP_HPLC (TFA) Purity by Area Report Result ≥0.1% Injection#1 = 0.07% Integration for: Largest Impurity Injection #2 = 0.08% (RRT1.04) Mean = 0.07% RP_HPLC (TFA) Purity by Area Report Result ≥0.1%Injection #1 = 0.12% Integration for: Largest Impurity Injection #2 =0.12% (RRT 0.96) Mean = 0.12% RP_HPLC (TFA) Purity by Area Report Result≥0.1% Injection #1 = 0.49% Integration for: Largest Impurity Injection#2 = 0.49% (RRT 1.11) Mean = 0.49% Total Impurities Information 5.6%

Example 10: Von Heijne (VH) Value Calculation

von Heijne values were calculated using a method adapted from Hessa etal., Recognition of transmembrane helices by the endoplasmic reticulumntranslocon, Nature: 433, 377-381 (2005). Briefly, each amino acid isassigned a fixed value, regardless of location along the polypeptidechain, according to the Table 18 below:

TABLE 18 von Heijne Score of various amino acids Amino Acid von HeijneScore I −0.6 L −0.5 Nle −0.5 $ −0.5 St −0.5 $e −0.5 $r8 −0.5 $r5 −0.5$s8 −0.5 $s5 −0.5 $er8 −0.5 F −0.3 V −0.3 Aib −0.1 M −0.1 C −0.1 Abu−0.1 Ac— 0 —NH₂ 0 A 0.1 a 0.1 W 0.3 T 0.5 Y 0.6 G 0.6 S 0.8 N 2 H 2 P2.2 Q 2.2 E 2.5 R 2.5 K 2.5 D 3.5

The von Heijne value (VH) for the polypeptide is then calculated as thesum total of values for all amino acids in the polypeptide. For example,a pentapeptide of the sequence Ac-AAAAA-NH₂ (SEQ ID NO: 5) would have aVH score of 5*(0.1)=0.5.

Example 11: Reverse-Phase HPLC Retention Time Determination

Peptides were analyzed by reverse-phase HPLC on a 100×2.1 mm Phenomenex2.6 micron, 100 Angstrom C18 column using the following mobile phasegradient at mom temperature:

Time Flow rate % A % B (min) mL/min) (0.1% TFA in water) (0.1% TFA inacetonitrile) 0 0.6 80 20 20.0 0.6 20 80 20.1 0.6 5 95 21.0 0.6 5 9521.1 0.6 80 20 21.2 0.6 5 95 21.5 0.6 5 95 21.8 0.6 80 20 23.5 0.6 80 20

In some embodiments, the retention time (RT) was then normalized to a0-100 scale by the following equation: RT=[RT_raw (fromabove)*3.317-0.534]*3.3333. In some embodiments, the retention timeswere not normalized.

What is claimed: 1-293. (canceled)
 294. An aqueous pharmaceuticalformulation in a unit dosage form comprising: (i) a peptidomimeticmacrocycle or a pharmaceutically acceptable salt thereof, wherein thepeptidomimetic macrocycle; (ii) a buffering agent; (iii) a stabilizingagent that is polysorbate 20; (iv) a tonicity agent that is trehalose;wherein the peptidomimetic macrocycle has a Formula I:

wherein each D, Xaa₅, Xaa₆, Xaa₈, Xaa₉, and E is independently an aminoacid; each E is independently selected from Ala, D-Ala, Aib, Sar, andSer; [D]_(v) is Leu₁-Thr₂; Xaa₃ is Phe; Xaa₇ is Trp; Xaa₁₀ is Leu; eachR₁ and R₂ is independently alkyl; L is a macrocycle-forming linker; R₇is —H; R₈ is —H; and w is an integer from 1-10.
 295. The aqueouspharmaceutical formulation of claim 294, wherein the peptidomimeticmacrocycle has a length value of from 14 to 20 amino acids.
 296. Theaqueous pharmaceutical formulation of claim 294, wherein thepeptidomimetic macrocycle has a von Heijne value of from 2 to
 9. 297.The aqueous pharmaceutical formulation of claim 294, wherein thepeptidomimetic macrocycle has a percent alanine content of from 15% to40%.
 298. The aqueous pharmaceutical formulation of claim 294, wherein afirst, second, third, fourth, fifth, or sixth C-terminal amino acid ofthe peptidomimetic macrocycle is hydrophobic.
 299. The aqueouspharmaceutical formulation of claim 294, wherein the peptidomimeticmacrocycle comprises an α-helix.
 300. The aqueous pharmaceuticalformulation of claim 294, wherein the pharmaceutically acceptable saltof the peptidomimetic macrocycle is a sodium, potassium, lithium,calcium, zinc, or magnesium salt.
 301. The aqueous pharmaceuticalformulation of claim 294, wherein total peptidomimetic degradationproducts formed in the aqueous pharmaceutical formulation is less than1.0% when stored at a temperature of 40° C. for a period of one month.302. The aqueous pharmaceutical formulation of claim 294, wherein anosmolarity of the aqueous pharmaceutical formulation is from about 250to about 1000 milliosmoles per kilogram.
 303. The aqueous pharmaceuticalformulation of claim 294, further comprising glucose, fructose,galactose, sucrose, lactose, maltose, or a mixture thereof.
 304. Theaqueous pharmaceutical formulation of claim 294, wherein the aqueouspharmaceutical formulation has a pH from about 6.0 to about 8.0. 305.The aqueous pharmaceutical formulation of claim 294, wherein the aqueouspharmaceutical formulation has a pH from about 4.0 to about 9.0. 306.The aqueous pharmaceutical formulation of claim 294, wherein thepeptidomimetic macrocycle has a molecular weight in the range of1800-2000 D.
 307. A method of making an aqueous pharmaceuticalformulation comprising adding greater than 15 mg/mL of a peptidomimeticmacrocycle or a pharmaceutically acceptable salt thereof to water or anaqueous solution, wherein the aqueous pharmaceutical formulationcomprises less than 2% w/v of any micelle forming agent.
 308. The methodof claim 307, wherein the peptidomimetic macrocycle is capable ofbinding to the MDM2 and/or MDMX proteins.
 309. The method of claim 307,comprising adding a sodium salt of the peptidomimetic macrocycle towater or an aqueous solution.
 311. The method of claim 307, furthercomprising adjusting the pH of the solution comprising the bufferingagent and the stabilizing agent during the addition of thepeptidomimetic macrocycle.
 312. The method of claim 307, furthercomprising filtration of the aqueous pharmaceutical formulation obtainedafter the addition of the peptidomimetic macrocycle to the aqueoussolution.
 313. The aqueous pharmaceutical formulation of claim 294,wherein the aqueous pharmaceutical formulation is suitable foradministration to a subject without dilution.